Novel micro-dystrophins and related methods of use

ABSTRACT

Nucleotide sequences including a micro-dystrophin gene are provided. The micro-dystrophin genes may be operatively linked to a regulatory cassette. Methods of treating a subject having, or at risk of developing, muscular dystrophy, sarcopenia, heart disease, or cachexia are also provided. The methods may include administering a pharmaceutical composition including the micro-dystrophin gene and a delivery vehicle to a subject. Further, the methods may include administering the pharmaceutical composition a subject having Duchenne muscular dystrophy or Becker muscular dystrophy.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation under 35 U.S.C. § 120 of co-pendingU.S. application Ser. No. 15/541,870 filed on Jul. 6, 2017, which is a35 U.S.C. § 371 National Phase Entry of the International ApplicationNo. PCT/US2016/013733 filed on Jan. 15, 2016, which designates the U.S.,and claims benefit under 35 U.S.C. 119(e) of U.S. Provisional PatentApplication No. 62/104,537, filed on Jan. 16, 2015, the contents ofwhich are incorporated herein by reference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant No. R01AG033610, awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 6, 2017 isnamed 034186-095700USPX.txt and is 141,620 bytes in size.

TECHNICAL FIELD

The present disclosure relates generally to micro-dystrophins. Thepresent disclosure also relates to methods of treating a subject havingmuscular dystrophy, sarcopenia, heart failure, or cachexia. The presentdisclosure also relates to methods of prophylactically treating asubject at risk of developing muscular dystrophy, sarcopenia, heartfailure, or cachexia. In particular, the methods may includeadministering a pharmaceutical composition including a micro-dystrophingene and a delivery vehicle to a subject. More particularly, the methodsmay include administering the pharmaceutical composition to a subjecthaving Duchenne muscular dystrophy or Becker muscular dystrophy.

BACKGROUND

Duchenne muscular dystrophy (DMD) is a recessively-inherited musclewasting disorder that affects approximately 1 in 3500 males. DMDpatients carry a mutation in the dystrophin gene that causes aberrantexpression or loss of expression of the dystrophin protein. DMD patientsexperience progressive wasting of skeletal muscles and cardiacdysfunction, which leads to loss of ambulation and premature death,primarily due to cardiac or respiratory failure. Unfortunately,currently available treatments are generally only able to slow thepathology of DMD. Accordingly, there is an urgent need for compositionsand methods for treating DMD.

SUMMARY OF THE INVENTION

The present disclosure is based, at least in part, on novelmicro-dystrophins, compositions thereof, and related methods of use.

In some embodiments of the present disclosure, the isolated and purifiednucleotide sequence, includes: (a) a micro-dystrophin gene encoding aprotein including: an amino-terminal actin-binding domain; aβ-dystroglycan binding domain; and a spectrin-like repeat domain,including at least four spectrin-like repeats, such that two of the atleast four spectrin-like repeats include a neuronal nitric oxidesynthase binding domain; and (b) a regulatory cassette.

In one embodiment, the at least four spectrin-like repeats includespectrin-like repeat 1 (SR1), spectrin-like repeat 16 (SR16),spectrin-like repeat 17 (SR17), and spectrin-like repeat 24 (SR24).

In another embodiment, the protein encoded by the micro-dystrophin genefurther includes at least a portion of a hinge domain.

In yet another embodiment, the hinge domain is selected from at leastone of a Hinge 1 domain, a Hinge 2 domain, a Hinge 3 domain, a Hinge 4domain, and a hinge-like domain.

In still another embodiment, the regulatory cassette is selected fromthe group consisting of a CK8 promoter and a cardiac troponin T (cTnT)promoter.

In one embodiment, the protein encoded by the micro-dystrophin gene hasbetween five spectrin-like repeats and eight spectrin-like repeats.

In another embodiment, the protein encoded by the micro-dystrophin genehas at least 80% sequence identity to the amino acid sequence of SEQ IDNO:4.

In yet another embodiment, the protein encoded by the micro-dystrophingene has at least 90% sequence identity to the amino acid sequence ofSEQ ID NO:4.

In still another embodiment, the protein encoded by the micro-dystrophingene has at least 80% sequence identity to the amino acid sequence ofSEQ ID NO:5.

In one embodiment, the protein encoded by the micro-dystrophin gene hasat least 90% sequence identity to the amino acid sequence of SEQ IDNO:5.

In another embodiment, the regulatory cassette is the CK8 promoter, andwherein the CK8 promoter has at least 80% sequence identity to thenucleic acid sequence of SEQ ID NO:19.

In yet another embodiment, the regulatory cassette is the CK8 promoter,and wherein the CK8 promoter has at least 90% sequence identity to thenucleic acid sequence of SEQ ID NO:19.

In still another embodiment, the regulatory cassette is the cTnTpromoter, and wherein the cTnT promoter has at least 80% sequenceidentity to the nucleic acid sequence of SEQ ID NO:1.

In one embodiment, the regulatory cassette is the cTnT promoter, andwherein the cTnT promoter has at least 90% sequence identity to thenucleic acid sequence of SEQ ID NO:1.

In certain embodiments of the present disclosure, the isolated andpurified nucleotide sequence, includes: a micro-dystrophin gene encodinga protein including: an amino-terminal actin-binding domain; and atleast two spectrin-like repeats that are directly coupled to each other,wherein the at least two spectrin-like repeats that are directly coupledto each other are selected from at least one of spectrin-like repeat 1directly coupled to spectrin-like repeat 2, spectrin-like repeat 2directly coupled to spectrin-like repeat 3, spectrin-like repeat 1directly coupled to spectrin-like repeat 16, spectrin-like repeat 17directly coupled to spectrin-like repeat 23, spectrin-like repeat 17directly coupled to spectrin-like repeat 24, and spectrin-like repeat 23directly coupled to spectrin-like repeat 24.

In certain other embodiments of the present disclosure, the isolated andpurified nucleotide sequence, includes: a micro-dystrophin gene encodinga protein including, in order: a Hinge 1 domain (H1); a spectrin-likerepeat 1 (SR1); a spectrin-like repeat 16 (SR16); a spectrin-like repeat17 (SR17); a spectrin-like repeat 24 (SR24); and a Hinge 4 domain (H4).

In one embodiment, the H1 is directly coupled to the SR1.

In another embodiment, the SR 1 is directly coupled to the SR16.

In yet another embodiment, the SR16 is directly coupled to the SR17.

In still another embodiment, the SR 17 is directly coupled to the SR24.

In another embodiment, the SR24 is directly coupled to the H4.

In yet another embodiment, the protein encoded by the micro-dystrophingene further includes, between the SR1 and the SR16, in order, aspectrin-like repeat 2 (SR2) and a spectrin-like repeat 3 (SR3).

In still another embodiment, the SR1 is directly coupled to the SR2 andthe SR2 is further coupled to the SR3.

In some embodiments of the present disclosure, the isolated and purifiednucleotide sequence, includes: a micro-dystrophin gene encoding aprotein including, in order: a Hinge 1 domain (H1); a spectrin-likerepeat 1 (SR1); a spectrin-like repeat 16 (SR16); a spectrin-like repeat17 (SR17); a spectrin-like repeat 23 (SR 23); a spectrin-like repeat 24(SR24); and a Hinge 4 domain (H4).

In one embodiment, the H1 is directly coupled to the SR1, the SR1 isdirectly coupled to the SR16, the SR16 is directly coupled to the SR17,the SR17 is directly coupled to the SR23, the SR23 is directly coupledto the SR24, and the SR24 is directly coupled to the H4.

In certain embodiments of the present disclosure, the pharmaceuticalcomposition, includes: an isolated and purified nucleotide sequencedescribed herein; and a delivery vehicle.

In one embodiment, the delivery vehicle includes a recombinantadeno-associated virus vector.

In another embodiment, the delivery vehicle expresses themicro-dystrophin gene, such that the protein encoded by themicro-dystrophin gene has at least 80% sequence identity to the aminoacid sequence of SEQ ID NO:4.

In yet another embodiment, the delivery vehicle expresses themicro-dystrophin gene, such that the protein encoded by themicro-dystrophin gene has at least 90% sequence identity to the aminoacid sequence of SEQ ID NO:4.

In still another embodiment, the delivery vehicle expresses themicro-dystrophin gene, such that the protein encoded by themicro-dystrophin gene has at least 80% sequence identity to the aminoacid sequence of SEQ ID NO:5.

In another embodiment, the delivery vehicle expresses themicro-dystrophin gene, such that the protein encoded by themicro-dystrophin gene has at least 90% sequence identity to the aminoacid sequence of SEQ ID NO:5.

In some embodiments of the present disclosure, the pharmaceuticalcompositions described herein include a regulatory cassette, such thatthe regulatory cassette is the CK8 promoter, and the CK8 promoter has atleast 80% sequence identity to the nucleic acid sequence of SEQ IDNO:19.

In certain embodiments of the present disclosure, the pharmaceuticalcompositions described herein include a regulatory cassette, such thatthe regulatory cassette is the CK8 promoter, and the CK8 promoter has atleast 90% sequence identity to the nucleic acid sequence of SEQ IDNO:19.

In some embodiments of the present disclosure, the pharmaceuticalcompositions described herein include a regulatory cassette, such thatthe regulatory cassette is the cTnT promoter, and the cTnT promoter hasat least 80% sequence identity to the nucleic acid sequence of SEQ IDNO:1.

In certain embodiments of the present disclosure, the pharmaceuticalcompositions described herein include a regulatory cassette, such thatthe regulatory cassette is the cTnT promoter, and the cTnT promoter hasat least 90% sequence identity to the nucleic acid sequence of SEQ IDNO:1.

In some embodiments of the present disclosure, the pharmaceuticalcomposition is configured to reduce a pathological effect or symptom ofa muscular dystrophy selected from at least one of myotonic musculardystrophy, Duchenne muscular dystrophy, Becker muscular dystrophy,limb-girdle muscular dystrophy, facioscapulohumeral muscular dystrophy,congenital muscular dystrophy, oculopharyngeal muscular dystrophy,distal muscular dystrophy, and Emery-Dreifuss muscular dystrophy.

In certain embodiments of the present disclosure, the pharmaceuticalcomposition is configured to reduce a pathological effect or symptom ofa muscular dystrophy selected from at least one of Duchenne musculardystrophy and Becker muscular dystrophy.

In some embodiments of the present disclosure, the pharmaceuticalcomposition is configured to reduce a pathological effect or symptom ofat least one of sarcopenia, heart disease, and cachexia.

In particular embodiments of the present disclosure, the pharmaceuticalcomposition, includes: a micro-dystrophin gene including the nucleicacid sequence of SEQ ID NO:16; and an adeno-associated virus (AAV)vector or a recombinant adeno-associated virus (rAAV) vector. In certainembodiments, the serotype of the AAV vector or the rAAV vector isselected from at least one of serotype 6, serotype 8, and serotype 9.

In some embodiments of the present disclosure, the pharmaceuticalcomposition, includes: a micro-dystrophin gene encoding a protein, suchthat the protein includes the amino acid sequence of SEQ ID NO:4; and anadeno-associated virus (AAV) vector or a recombinant adeno-associatedvirus (rAAV) vector. In certain embodiments, the serotype of the AAVvector or the rAAV vector is selected from at least one of serotype 6,serotype 8, and serotype 9.

In certain embodiments of the present disclosure, the pharmaceuticalcomposition, includes: a micro-dystrophin gene including the nucleicacid sequence of SEQ ID NO:18; and an adeno-associated virus (AAV)vector or a recombinant adeno-associated virus (rAAV) vector. In someembodiments, the serotype of the AAV vector or the rAAV vector isselected from at least one of serotype 6, serotype 8, and serotype 9.

In particular embodiments of the present disclosure, the pharmaceuticalcomposition, includes: a micro-dystrophin gene encoding a protein, suchthat the protein includes the amino acid sequence of SEQ ID NO:5; and anadeno-associated virus (AAV) vector or a recombinant adeno-associatedvirus (rAAV) vector. In some embodiments, the serotype of the AAV vectoror the rAAV vector is selected from at least one of serotype 6, serotype8, and serotype 9.

In some embodiments of the present disclosure, the pharmaceuticalcompositions suitable for use in the treatment or prophylactic treatmentof muscular dystrophy, include: a micro-dystrophin gene including thenucleic acid sequence of SEQ ID NO:16 or SEQ ID NO:18; and anadeno-associated virus (AAV) vector or a recombinant adeno-associatedvirus (rAAV) vector, such that the serotype of the AAV vector or therAAV vector is selected from at least one of serotype 6, serotype 8, andserotype 9.

In certain embodiments of the present disclosure, the pharmaceuticalcompositions suitable for the treatment or prophylactic treatment ofmuscular dystrophy, include: a micro-dystrophin gene including thenucleic acid sequence of SEQ ID NO:16 or SEQ ID NO:18; and anadeno-associated virus (AAV) vector or a recombinant adeno-associatedvirus (rAAV) vector, such that the serotype of the AAV vector or therAAV vector is selected from at least one of serotype 6, serotype 8, andserotype 9.

In particular embodiments of the present disclosure, the methods fortreating a subject having muscular dystrophy, include: administering tothe subject a therapeutically effective amount of a pharmaceuticalcomposition including a micro-dystrophin gene operably coupled to aregulatory cassette.

In one embodiment, the regulatory cassette is selected from the groupconsisting of a CK8 promoter and a cardiac troponin T (cTnT) promoter.

In another embodiment, the regulatory cassette is configured to expressthe micro-dystrophin gene such that a level of expression of themicro-dystrophin gene is at least 100-fold higher in striated musclecells than the level of expression of the micro-dystrophin gene innon-muscle cells.

In certain embodiments of the present disclosure, the pharmaceuticalcompositions described herein further include a recombinantadeno-associated virus vector configured to express the micro-dystrophingene in the subject.

In one embodiment, the micro-dystrophin gene encodes a protein having atleast 80% sequence identity to the amino acid sequence of SEQ ID NO:4.

In another embodiment, the micro-dystrophin gene encodes a proteinhaving at least 90% sequence identity to the amino acid sequence of SEQID NO:4.

In yet another embodiment, the micro-dystrophin gene encodes a proteinhaving at least 80% sequence identity to the amino acid sequence of SEQID NO:5.

In still another embodiment, the micro-dystrophin gene encodes a proteinhaving at least 90% sequence identity to the amino acid sequence of SEQID NO:5.

In one embodiment, the regulatory cassette is the CK8 promoter, and theCK8 promoter has at least 80% sequence identity to the nucleic acidsequence of SEQ ID NO:19.

In another embodiment, the regulatory cassette is the CK8 promoter, andthe CK8 promoter has at least 90% sequence identity to the nucleic acidsequence of SEQ ID NO:19.

In yet another embodiment, the regulatory cassette is the cTnT promoter,and the cTnT promoter has at least 80% sequence identity to the nucleicacid sequence of SEQ ID NO:1.

In still another embodiment, the regulatory cassette is the cTnTpromoter, and the cTnT promoter has at least 90% sequence identity tothe nucleic acid sequence of SEQ ID NO:1.

In one embodiment, the micro-dystrophin gene expresses amicro-dystrophin protein in one or more muscles of the subject such thatcontractility of the one or more muscles is enhanced.

In another embodiment, the micro-dystrophin gene expresses amicro-dystrophin protein in one or more skeletal muscles of the subjectsuch that a specific-force generating capacity of at least one of theone or more skeletal muscles is increased to within at least 40% of anormal specific-force generating capacity.

In yet another embodiment, the micro-dystrophin gene expresses amicro-dystrophin protein in one or more cardiac muscles of the subjectsuch that a baseline end-diastolic volume defect is restored to withinat least 40% of a normal end-diastolic volume.

In still another embodiment, the micro-dystrophin gene expresses amicro-dystrophin protein such that localization of the neuronal nitricoxide synthase to the dystrophin-glycoprotein complex is enhanced in thesubject.

In some embodiments, the muscular dystrophy is selected from at leastone of myotonic muscular dystrophy, Duchenne muscular dystrophy, Beckermuscular dystrophy, limb-girdle muscular dystrophy, facioscapulohumeralmuscular dystrophy, congenital muscular dystrophy, oculopharyngealmuscular dystrophy, distal muscular dystrophy, and Emery-Dreifussmuscular dystrophy.

In certain embodiments, the muscular dystrophy is selected from at leastone of Duchenne muscular dystrophy and Becker muscular dystrophy.

In some embodiments of the present disclosure, the pharmaceuticalcomposition reduces a pathological effect or symptom of the musculardystrophy.

In particular embodiments, the pathological effect or symptom of themuscular dystrophy is selected from at least one of muscle pain, muscleweakness, muscle fatigue, muscle atrophy, fibrosis, inflammation,increase in average myofiber diameter in skeletal muscle,cardiomyopathy, reduced 6-minute walk test time, loss of ambulation, andcardiac pump failure.

In some embodiments, the methods described herein include identifyingthe subject having the muscular dystrophy.

In certain embodiments, the subject is a mammal.

In particular embodiments, the subject is a human.

In some embodiments of the present disclosure, the methods forprophylactically treating a subject at risk of developing musculardystrophy, include administering to the subject a therapeuticallyeffective amount of a pharmaceutical composition including amicro-dystrophin gene operably coupled to a regulatory cassette.

In one embodiment, the regulatory cassette is selected from the groupconsisting of a CK8 promoter and a cardiac troponin T (cTnT) promoter.

In further embodiments, the regulatory cassette is configured to expressthe micro-dystrophin gene such that a level of expression of themicro-dystrophin gene is at least 100-fold higher in striated musclecells than the level of expression of the micro-dystrophin gene innon-muscle cells.

In particular embodiments, the pharmaceutical composition furtherincludes a recombinant adeno-associated virus vector configured toexpress the micro-dystrophin gene in the subject.

In certain embodiments, the micro-dystrophin gene encodes a proteinhaving at least 80% sequence identity to the amino acid sequence of SEQID NO:4.

In another embodiment, the micro-dystrophin gene encodes a proteinhaving at least 90% sequence identity to the amino acid sequence of SEQID NO:4.

In some embodiments, the micro-dystrophin gene encodes a protein havingat least 80% sequence identity to the amino acid sequence of SEQ IDNO:5.

In yet another embodiment, the micro-dystrophin gene encodes a proteinhaving at least 90% sequence identity to the amino acid sequence of SEQID NO:5.

In certain embodiments, the regulatory cassette is the CK8 promoter, andthe CK8 promoter has at least 80% sequence identity to the nucleic acidsequence of SEQ ID NO:19.

In another embodiment, the regulatory cassette is the CK8 promoter, andthe CK8 promoter has at least 90% sequence identity to the nucleic acidsequence of SEQ ID NO:19.

In yet another embodiment, the regulatory cassette is the cTnT promoter,and the cTnT promoter has at least 80% sequence identity to the nucleicacid sequence of SEQ ID NO:1.

In still another embodiment, the regulatory cassette is the cTnTpromoter, and the cTnT promoter has at least 90% sequence identity tothe nucleic acid sequence of SEQ ID NO:1.

In particular embodiments, the micro-dystrophin gene expresses amicro-dystrophin protein in one or more muscles of the subject such thatcontractility of the one or more muscles is enhanced.

In another embodiment, the micro-dystrophin gene expresses amicro-dystrophin protein in one or more skeletal muscles of the subjectsuch that a specific-force generating capacity of at least one of theone or more skeletal muscles is increased to within at least 40% of anormal specific-force generating capacity.

In some embodiments, the micro-dystrophin gene expresses amicro-dystrophin protein in one or more cardiac muscles of the subjectsuch that a baseline end-diastolic volume defect is restored to withinat least 40% of a normal end-diastolic volume.

In certain embodiments, the micro-dystrophin gene expresses amicro-dystrophin protein such that localization of the neuronal nitricoxide synthase to the dystrophin-glycoprotein complex is enhanced in thesubject.

In particular embodiments, the muscular dystrophy is selected from atleast one of myotonic muscular dystrophy, Duchenne muscular dystrophy,Becker muscular dystrophy, limb-girdle muscular dystrophy,facioscapulohumeral muscular dystrophy, congenital muscular dystrophy,oculopharyngeal muscular dystrophy, distal muscular dystrophy, andEmery-Dreifuss muscular dystrophy.

In some embodiments, the muscular dystrophy is selected from at leastone of Duchenne muscular dystrophy and Becker muscular dystrophy.

In certain embodiments, the pharmaceutical compositions described hereinreduce a risk of developing a pathological effect or symptom of themuscular dystrophy.

In one embodiment, the pathological effect or symptom of the musculardystrophy is selected from at least one of muscle pain, muscle weakness,muscle fatigue, muscle atrophy, fibrosis, inflammation, increase inaverage myofiber diameter in skeletal muscle, cardiomyopathy, reduced6-minute walk test time, loss of ambulation, and cardiac pump failure.

In some embodiments of the present disclosure, the methods describedherein further include identifying the subject at risk of developing themuscular dystrophy.

In one embodiment, the subject is a mammal.

In another embodiment, the subject is a human.

Other features and advantages of the disclosure will be apparent fromthe following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The embodiments disclosed herein will become more fully apparent fromthe following description and appended claims, taken in conjunction withthe accompanying drawings.

FIG. 1A depicts protein structure diagrams of embodiments of truncateddystrophin constructs as disclosed herein. NT, amino terminal domain; H,hinge; R, spectrin-like repeat; nNOS BD, neuronal nitric oxide synthasebinding domain; CR, cysteine-rich domain; CT, carboxyl terminal domain;Syn, syntrophin binding domain; Db BD, dystrobrevin binding domain; theunlabeled region marks 20-amino acids between R15 and R16; aa, aminoacid; and kDa, kilodalton.

FIG. 1B is a Western blot illustrating the results of injectingdystrophic mdx^(4cv) mice with 5×10¹⁰ vector genomes (vg) ofrAAV/CMV-μDys into one tibialis anterior (TA) muscle while, thecontralateral muscle served as an internal, untreated control.Expression of all tested constructs was verified at 4 weeks aftertreatment by Western blot analysis of TA muscle lysates, along with wildtype and untreated mdx^(4cv) controls. Glyceraldehyde-3-phosphatedehydrogenase (GAPDH) served as an internal loading control.

FIGS. 1C and 1D are graphs depicting quantification of myofibers from TAcross sections for dystrophin expression and central nucleation at 4 or12 weeks post-treatment, respectively (N=3-5 per cohort for each timepoint, mean±S.E.M.). μDysH3 served as a comparative gauge ofperformance. μDys6 and μDys7 were too large to be cloned intoAAV-expression vectors using the ubiquitous cytomegalovirus (CMV)promoter, consequently, the CMV promoter was replaced with themyogenic-specific CK8 promoter to allow efficient packaging and in vivoevaluation. Accordingly, μDysH3 was re-evaluated with the CK8 regulatoryexpression cassette. Characters denote significance from wild type mice.*P<0.05, **P<0.01, ***P<0.001, #P<0.0001.

FIG. 2 is a series of micrographs depicting representative gastrocnemiuscross sections at six months post-treatment. Dystrophin and DAPI-stainednuclei are shown in the left column, β-dystroglycan and DAPI are shownin the middle column, and neuronal nitric oxide synthase (nNOS) is shownin the right column, as indicated. Each row depicts representativeresults from cohorts of wild type, treated mdx^(4cv), and untreatedmdx^(4cv) mice. Scale bar, 200 μm. Recruitment of dystrophinglycoprotein complex (DGC) members is generally dependent on bindingdomains within μDys constructs. Dystrophic mdx^(4cv) mice were injectedretro-orbitally with 1×10¹³ vg of rAAV6/CK8-μDys at 14 days of age.Three and six months post-treatment, skeletal muscles were immunostainedfor DGC members.

FIGS. 3A-3D are graphs depicting evaluation of systemic treatment at 3months post-treatment. Gastrocnemius muscles (FIGS. 3A and 3B) anddiaphragm muscles (FIGS. 3C and 3D) were evaluated to determine theperformance of novel μDys constructs. Muscle cross sections werequantified for dystrophin expression and centrally nucleated myofibers.Levels of myofibers exhibiting dystrophin expression and/or exhibitingcentral nucleation are represented as percentages (FIGS. 3A and 3C).Specific force generation was measured in situ for gastrocnemius (FIG.3B) and in vitro for diaphragm strips (FIG. 3D). The n value for eachcohort is listed in columns of FIG. 3D. For non-bracketed characters,*P<0.05, **P<0.01, ***P<0.001 from wild type. {circumflex over( )}P<0.05, {circumflex over ( )}{circumflex over ( )}P<0.01,{circumflex over ( )}{circumflex over ( )}{circumflex over ( )}P<0.001from μDys2-treated mice. #P<0.05, ##P<0.01, ###P<0.001 fromμDys5-treated mice.

FIGS. 4A-4D are graphs depicting evaluation of systemic treatment at 6months post-treatment. Gastrocnemius muscles (FIGS. 4A and 4B) anddiaphragm muscles (FIGS. 4C and 4D) were evaluated as described in FIGS.3A-3D. The n value for each cohort is listed in columns of FIG. 4D.*P<0.05, **P<0.01, ***P<0.001 from wild type. {circumflex over( )}P<0.05, {circumflex over ( )}{circumflex over ( )}P<0.01,{circumflex over ( )}{circumflex over ( )}{circumflex over ( )}P<0.001from μDys2-treated mice. #P<0.05, ##P<0.01, ###P<0.001 fromμDys7-treated mice.

FIGS. 5A and 5B are graphs depicting the extent of sarcolemmalprotection from eccentric contraction in skeletal muscles. Systemicallytreated mice, as described in FIGS. 2 and 4A-4D, were subjected toeccentric contractions of increasing length. Gastrocnemius (FIG. 5A) anddiaphragm strips (FIG. 5B) were measured for the maximum isometric forcegenerated prior to an eccentric contraction. During stimulatingcontractions, muscles were lengthened at a defined distance beyond theiroptimum fiber lengths. Distances are reported as percentage beyondoptimal fiber length (L_(o)). *P<0.05, ***P<0.001, ****P<0.0001 fromwild type at 45% beyond L_(o). {circumflex over ( )}{circumflex over( )}{circumflex over ( )}P<0.001, {circumflex over ( )}{circumflex over( )}P<0.0001 from μDys2-treated mice at 45% beyond L_(o). ∇∇∇∇P<0.0001from μDys7-treated mice at 45% beyond L_(o).

FIG. 6 is a series of micrographs illustrating that systemically testednovel μDys constructs do not induce ringbinden phenotype in skeletalmuscle. Dystrophic mdx^(4cv) mice were injected retro-orbitally with1×10¹³ vg at 14 days of age. Six months post-treatment, cross sectionsof gastrocnemius muscles were immunostained for dystrophin, DAPI, andα-sarcomeric actin. One representative section is shown from cohorts ofwild type (panel “a”) and mdx^(4cv) treated with μDysH3 (panel “b”),μDys1 (panel “c”), μDys2 (panel “d”), μDys5 (panel “e”), μDys6 (panel“f”), μDys7 (panel “g”), or untreated mdx^(4cv) mice (panel “h”).Gastrocnemius from transgenic mice expressing ΔR4-R23/ΔCT (see Harper,S. Q., et al., Nature Medicine 8, 253-261, (2002)) on mdx^(4cv)background (panel “i”) was also immunostained as a positive control.Arrowheads mark examples of ringbinden formation around myofibers. Scalebar, 50 μm.

FIG. 7 depicts protein structure diagrams of embodiments of novelmicro-dystrophin constructs as disclosed herein. The top proteinstructure diagram is of full-length dystrophin showing many of the knownfunctional domains: NT, amino terminal actin-binding domain; H, hinge;R, spectrin-like repeat; nNOS BD, neuronal nitric oxide synthase bindingdomain; CR, cysteine-rich domain; CT, carboxyl terminal domain; Dg BD,dystroglycan binding domain; Syn, syntrophin binding domain; Db BD,dystrobrevin binding domain; and the unlabeled region marks 20-aminoacids between R15 and R16. The WW domain is within Hinge 4. On the leftare shown the micro-dystrophin protein structures, with the designatedname to the left of the protein structure diagram, and the domainstructure listed to the right of the schematic diagram.

FIG. 8 is two graphs depicting left ventricle (LV) ejection fraction at2 weeks (left) and 3 weeks (right) for untreated (UN; n=S) vs. low (L;n=3) or high (H; n=3) dose of AAV6-L48Q.

FIG. 9 is an anti-cTnC Western blot for AAV6-L48Q cTnC injected mousecardiac tissue (left) and uninjected control (right), as indicated

FIG. 10A is a Western blot for RI, with GAPDH as a loading control.

FIG. 10B is a Western blot for R2, with GAPDH as a loading control.

FIG. 10C is a graph depicting HPLC of transfected cardiomyocytes [dATP].

FIG. 11 is two graphs. The graph at the left depicts the percentagefractional shortening (FS) increase in R1R2 over-expressing mice vs.control littermates. The graph at the right depicts the change in leftventricular inner diameter (LVID) in R1R2 over-expressing mice vs.control littermates. d-diastole, s-systole.

FIG. 12A depicts mouse aortic smooth muscle contraction traces with ATPand dATP.

FIG. 12B is a graph depicting a summary of the data in FIG. 12A.

FIG. 13A depicts Western blots for R1 and R2.

FIG. 13B depicts α-tubulin as a loading control for the Western blots ofFIG. 13A.

FIG. 14 shows preliminary Western blot evidence for the expressionlevels of RI and R2 subunits in the skeletal muscle, lung, and heart ofrAAV6-R1R2^(cTnT455) injected (4.5×10¹³) mice and control mice (panel“A”). FIG. 14 also provides data for heart tissue from non-injected(panel “B”) vs. AAV6-alkaline phosphatase (panel “C”) injected mice (seeRafael, J. A., et al., The Journal of Cell Biology 134, 93-102 (1996))after 20 months, suggesting AAV6-R1R2^(cTnT455) may provide stable,long-term R1R2 over-expression.

FIG. 15 is a graph showing the effect of 1.5×10¹³, 4.5×10¹³, and1.35×10¹⁴ rAAV6-RIR2^(cTnT455) vector genomes or saline (control)injected systemically over an approximate 10-fold range into 3 month oldmice (n=6 per group) on LV function.

FIG. 16 is two graphs showing the change in fractional shortening inrats given direct cardiac injections of rAAV6-R1R2 on the fifth daypost-infarct as measured by echocardiography in comparison withuntreated infarct rats and untreated sham-operated rats.

FIG. 17 is a graph showing the in vitro Neely working heart measurementsof the rat hearts assessed in FIG. 16. Power on the y-axis is given inunits of g·cm/min. A loss of pre-load responsiveness of hearts (heartfailure) that have been infarcted (no treatment) and a recovery ofpre-load responsiveness of the infarcted hearts receiving the vectors tothe level of control, uninfarcted hearts were observed, therebydemonstrating a restoration of cardiac function.

FIG. 18 illustrates miniaturization of human-cTnT (Enh+Promoter)regulatory cassettes based on deleting sequences hypothesized to haverelatively low activities.

FIG. 19 illustrates transcription tests of the FIG. 18 deletionsrelative to a native human-cTnT enhancer/promoter; the 320 bp versionretains ˜95% of the activity.

FIG. 20 illustrates increased activity of human-cTnT455 via adding asecond miniaturized enhancer compared to the native enhancer/promoter,the 320 bp version, and to a Chicken cTnT promoter/enhancer (seeAmerican Journal of Physiology—Cell Physiology 280, C556-0564 (2004)).

FIG. 21 is a series of schematic illustrations of the structure ofdystrophin spectrin-like repeats and the juxtaposition with hingedomains. Top left, interdigitated folding of individual spectrin-likerepeats is illustrated to show how 3 adjacent repeats can fold together,with the different alpha-helical segments highlighted (a, b, c; a′, b′,c′; and a″, b″, c″ representing the helical domains of the threedifferent spectrin-like repeats). Top right, in native dystrophin andutrophin, some spectrin-like repeats are separated by hinge domains thatdisrupt the normal interdigitated folding of adjacent spectrin-likerepeats. Shown at the top right is the folding pattern of Spectrin-likerepeats 18, 19, and 20 and their separation by Hinge 3. Middle left,Optimized mini- and micro-dystrophins typically display maximalfunctional activity when the spectrin-like repeats domains are arrangedin such a way as to preserve normal folding patterns; this normalfolding is disrupted when non-integral units of spectrin-like repeatsare present in a mini- or micro-dystrophin proteins, such as when anatural occurring deletion that removes whole exons occurs in a Beckermuscular dystrophy patient. This latter situation is illustrated in theschematic illustration at the middle right, which represents thepredicted structure of the junctional domain of dystrophin from apatient with a genomic deletion removing exons 17-48. The bottomschematic illustrations show the folding pattern predicted in the μDysH2(left) and μDysH3 (right) proteins, and also illustrate theunpredictable nature of the functional activity of miniaturizeddystrophin proteins. While μDysH2 and μDysH3 have similar foldingpatterns, μDysH2 leads to ringbinden when expressed in mdx mouseskeletal muscles, whereas μDysH3 does not lead to ringbinden (see Banks,G. B., et al., PLoS Genetics 6, e1000958, (2010)).

FIG. 22 is an image of an mdx^(4cv) mouse muscle cryosection that wasstained for dystrophin expression using an anti-dystrophin antibody.

DETAILED DESCRIPTION

The present disclosure features compositions and methods for treatingDuchenne muscular dystrophy (DMD). More particularly, the presentdisclosure relates to methods for producing mini-dystrophin proteins fortreating a subject having muscular dystrophy, DMD, sarcopenia, heartfailure, and/or cachexia. As described in detail below, the presentdisclosure is based, at least in part, on the unexpected discovery thatmini-dystrophin proteins comprising specific combinations of proteindomains (e.g., a mini-dystrophin protein including an N-terminal domain,H1 domain, SR1 domain, SR16 domain, SR17 domain, SR 23 domain, SR24domain, H4 domain, and CR domain) from the dystrophin protein are ableto restore dystrophin function to levels sufficient to treat musculardystrophy, DMD, sarcopenia, heart failure, and/or cachexia.

Duchenne muscular dystrophy (DMD) is a recessively-inherited musclewasting disorder afflicting approximately 1 in 3500 males. DMD patientscarry a mutation in the dystrophin gene, resulting in aberrant or absentexpression of the dystrophin protein. DMD patients experienceprogressive wasting of skeletal muscles and cardiac dysfunction, leadingto loss of ambulation and premature death, primarily due to cardiac orrespiratory failure. Current available treatments are generally onlyable to slow the pathology of DMD (see Emery, A. E. H. and Muntoni, F.,Duchenne Muscular Dystrophy, Third Edition (Oxford University Press,2003)). Gene therapy approaches for DMD have been demonstrated indystrophic animal models by either directly targeting a class ofmutations, as with exon skipping, or replacing the mutated gene withviral-vector mediated delivery (see Koo, T. and Wood, M. J. Human GeneTherapy 24, (2013); Benedetti, S., et al., The FEBS Journal 280,4263-4280, (2013); and Seto, J. T., et al., Current Gene Therapy 12,139-151 (2012)). Recombinant adeno-associated virus (rAAV) vectors are apotential vehicle for gene therapy, being already tested in clinicaltrials for both DMD and limb-girdle muscular dystrophies (see Mendell,J. R., et al., The New England Journal of Medicine 363, 1429-1437,(2010); Mendell, J. R., et al., Annals of Neurology 68, 629-638 (2010);and Herson, S., et al., Brain: A Journal of Neurology 135, 483-492,(2012)). Several serotypes of adeno-associated virus (AAV) demonstrate ahigh degree of tropism for striated muscles (see Seto, J. T., et al.,Current Gene Therapy 12, 139-151 (2012)).

Pre-clinical studies designing and testing newer generations oftherapeutic constructs for DMD can be confined by the approximately 4.9kb size of a single-stranded rAAV vector genome (see Dong, B., et al.,Molecular Therapy: The Journal of the American Society of Gene Therapy18, 87-92, (2010) and Wu, Z., et al., Molecular Therapy: The Journal ofthe American Society of Gene Therapy 18, 80-86, (2010)). Packaging theentire approximately 13.9 kb cDNA of the muscle-specific isoform ofdystrophin into a single rAAV capsid cannot be achieved, accordingly,miniaturized, synthetic versions of the muscle-specific isoform ofdystrophin cDNA may be used. Although in vivo recombination of two andthree rAAV vector genomes has been demonstrated to deliver a mini- orfull-length dystrophin coding sequence (see, Odom, G. L., et al.,Molecular Therapy: The Journal of the American Society of Gene Therapy19, 36-45, (2011); Lostal, W., et al., Human Gene Therapy, (2014); andKoo, T., et al., Human Gene Therapy 25, 98-108, (2014)), the efficiencyof delivering multiple vectors for reconstituting full-length dystrophinmay be suboptimal and can increase the overall dose of viral capsidproteins needed for delivering vectors. However, beneficialrAAV-mediated gene therapy has been achieved using rationally-designedminiature versions of the dystrophin cDNA based in part on mRNAexpressed in mild Becker muscular dystrophy patients carrying in-framedeletions within the gene (see Beggs, A. H., et al., American Journal ofHuman Genetics 49, 54-67 (1991); Koenig, M., et al., American Journal ofHuman Genetics 45, 498-506 (1989); Goldberg, L. R., et al., Annals ofNeurology 44, 971-976, (1998); and England, S. B., et al., Nature 343,180-182 (1990)). Studies in transgenic and vector treated dystrophicmice expressing various dystrophin truncations have identified severalelements of the dystrophin gene that may be present in a functionalmicro-dystrophin (μDys) (see Harper, S. Q., et al., Nature Medicine 8,253-261, (2002)).

The full-length striated muscle isoform of dystrophin can play a role intransmitting contractile force through the sarcolemma and out to theextracellular matrix. In addition to maintaining the mechanical linkbetween the intracellular cytoskeleton and the membrane bound dystrophinglycoprotein complex (DGC), dystrophin can also be a scaffold forsignaling proteins (see Ozawa, E. in Myology (ed. Franzini-Armstrong CEngel A) 455-470 (McGraw-Hill, 2004); Winder, S. J. Journal of MuscleResearch and Cell Motility 18, 617-629 (1997); and Campbell, K. P. andKahl, S. D. Nature 338, 259-262, (1989)). The amino-terminal domain ofdystrophin can bind to F-actin filaments of the intracellularcytoskeleton (see Way, M., et al., FEBS Letters 301, 243-245 (1992);Hemmings, L., et al., The Journal of Cell Biology 116, 1369-1380 (1992);Fabbrizio, E., et al., Biochemistry 32, 10457-10463 (1993); and Pavalko,F. M. and Otey, C. A. Proceedings of the Society for ExperimentalBiology and Medicine 205, 282-293 (1994)). The middle, rod domain is thelargest and is composed of 24 spectrin-like repeats (SRs) that areflanked and interspersed with at least four hinge sub-domains. The roddomain can give dystrophin elasticity and flexibility for maintainingthe integrity of the sarcolemma during muscle contractility (see Winder,S. J. Journal of Muscle Research and Cell Motility 18, 617-629 (1997)).Various SRs provide unique regions that can serve as additional bindingsites for the intracellular cytoskeleton, the sarcolemma, as well asmembers of the DGC (see Rybakova, I. N., et al., The Journal of CellBiology 135, 661-672 (1996); Warner, L. E., et al., Human MolecularGenetics 11, 1095-1105 (2002); Metzinger, L., et al., Human MolecularGenetics 6, 1185-1191 (1997); Lai, Y., et al., The Journal of ClinicalInvestigation 119, 624-635, (2009)). In particular, the cysteine-richdomain and the adjacent Hinge 4 region form the β-dystroglycan bindingdomain (Dg BD) (see Blake, D. J., et al., Physiological Reviews 82,291-329, (2002); Ishikawa-Sakurai, M., et al., Human Molecular Genetics13, 693-702, (2004)), while the carboxy-terminal domain is a scaffoldfor additional DGC components (see Abmayr S, in Molecular Mechanisms ofMuscular Dystrophies (ed. Winder, S. J.) 14-34 (Landes Biosciences,2006)).

Partially functional micro-dystrophins can improve the dystrophicpathology in striated muscle by protecting the sarcolemma fromcontraction-induced injury and increasing the capacity to generateforce. These parameters can be achieved by binding to F-actin filamentsand β-dystroglycan through the amino-terminal domain and the Dg BD (seeHarper, S. Q., et al., Nature Medicine 8, 253-261, (2002); Warner, L.E., et al., Human Molecular Genetics 11, 1095-1105 (2002); Cox, G. A.,et al., Nature Genetics 8, 333-339, (1994); Greenberg, D. S., et al.,Nature Genetics 8, 340-344, (1994); Gardner, K. L., et al., Gene Therapy13, 744-751, (2006); Corrado, K., et al., The Journal of Cell Biology134, 873-884 (1996); and Rafael, J. A., et al., The Journal of CellBiology 134, 93-102 (1996)). Without being bound by any one particulartheory, prior studies indicate these two domains must be connected by atleast four SRs from the central rod domain, but there are numerous waysin which miniaturized dystrophins containing at least four SRs can beconstructed. While some combinations of SRs have been shown to improvethe dystrophic pathophysiology, other combinations have not yieldedproteins with significant functional capacity (see Harper, S. Q., etal., Nature Medicine 8, 253-261, (2002) and Abmayr S, in MolecularMechanisms of Muscular Dystrophies (ed. Winder, S. J.) 14-34 (LandesBiosciences, 2006)). Selection of specific SRs in μDys design canrestore additional DGC components to the sarcolemma. Neuronal nitricoxide synthase (nNOS) is a signaling protein that can be involved invasodilation in response to muscle contractile activity (see Stamler, J.S. and Meissner, G. Physiological Reviews 81, 209-237 (2001); Brenman,J. E., et al., Cell 82, 743-752 (1995); Kobayashi, Y. M., et al., Nature456, 511-515, (2008); and Torelli, S., et al., Neuropathology andApplied Neurobiology 30, 540-545, (2004)), and the presence of SRs 16and 17 can be involved in proper association of nNOS with the DGC (see28 Lai, Y. et al., The Journal of Clinical Investigation 119, 624-635,(2009) and Lai, Y., et al., Proceedings of the National Academy ofSciences of the United States of America 110, 525-530, (2013)).

Sequences within spectrin-like repeats 20-24 as well as Hinge 4 can playa role in proper association of dystrophin with microtubules, which canbe important for maintaining the intracellular architecture and torqueproduction in skeletal muscle (see Prins, K. W. et al., The Journal ofCell Biology 186, 363-369, (2009) and Belanto, J. J., et al.,Proceedings of the National Academy of Sciences of the United States ofAmerica 111, 5723-5728, (2014)). Nonetheless, the carboxy-terminaldomain and most of the SR domains have been found dispensable withoutseverely compromising the health of striated muscles (see McCabe, E. R.,et al., The Journal of Clinical Investigation 83, 95-99, (1989);Crawford, G. E., et al., The Journal of Cell Biology 150, 1399-1410(2000); and Dunckley, M. G., et al., FEBS Letters 296, 128-134 (1992)).

Several of the best micro-dystrophins tested to date can protect musclesfrom contraction-induced injury and restore some, but generally not all,of the specific force generating capacity to dystrophic mouse and caninemodels for DMD (see Seto, J. T., et al., Current Gene Therapy 12,139-151 (2012) and Wang, Z., et al., Frontiers in Microbiology 2, 201,(2011)). Other micro-dystrophins carrying different combinations of SRsand hinges may function less well in dystrophic muscles, and the reasonsfor differences in functionality are not clear. However, without beingbound by any one particular theory, they may relate to effects onmicro-dystrophin elasticity, folding, stability, and the ability toassemble sub-portions of the DGC without steric hindrance.

The present disclosure relates generally to micro-dystrophins. Themicro-dystrophins may be operatively linked to a regulatory cassette.The present disclosure also relates to methods of treating a subjecthaving muscular dystrophy, sarcopenia, heart failure, or cachexia.Further, the present disclosure relates to methods of prophylacticallytreating a subject at risk of developing muscular dystrophy, sarcopenia,heart failure, or cachexia. The methods for treating a subject having,or at risk of developing, muscular dystrophy, sarcopenia, heart failure,or cachexia may comprise administering a pharmaceutical compositionincluding a micro-dystrophin gene and a delivery vehicle to the subject.

It will be readily understood that the embodiments, as generallydescribed herein, are exemplary. The following more detailed descriptionof various embodiments is not intended to limit the scope of the presentdisclosure, but is merely representative of various embodiments.Moreover, the order of the steps or actions of the methods disclosedherein may be changed by those skilled in the art without departing fromthe scope of the present disclosure. In other words, unless a specificorder of steps or actions is required for proper operation of theembodiment, the order or use of specific steps or actions may bemodified.

Unless specifically defined otherwise, the technical terms, as usedherein, have their normal meaning as understood in the art. Thefollowing terms are specifically defined with examples for the sake ofclarity.

As used herein, “peptide” and “polypeptide” may be used in theirbroadest senses to refer to a sequence of subunit amino acids. Thepeptides or polypeptides of the disclosure may comprise L-amino acids,D-amino acids (which can be resistant to L-amino acid-specific proteasesin vivo), or a combination of D- and L-amino acids. The terms peptideand polypeptide can be used interchangeably. The peptides andpolypeptides described herein may be chemically synthesized orrecombinantly expressed. The peptides and polypeptides may be linked toany other moiety as deemed useful for a given purpose. Such linkage cancomprise covalent linkages or non-covalent linkages as is understood bythose of skill in the art.

Amino acid residues as disclosed herein can be modified by conservativesubstitutions to maintain, or substantially maintain, overallpolypeptide structure and/or function. As used herein, “conservativeamino acid substitution” indicates that: hydrophobic amino acids (i.e.,Ala, Cys, Gly, Pro, Met, Val, Ile, and Leu) can be substituted withother hydrophobic amino acids; hydrophobic amino acids with bulky sidechains (i.e., Phe, Tyr, and Trp) can be substituted with otherhydrophobic amino acids with bulky side chains; amino acids withpositively charged side chains (i.e., Arg, His, and Lys) can besubstituted with other amino acids with positively charged side chains;amino acids with negatively charged side chains (i.e., Asp and Glu) canbe substituted with other amino acids with negatively charged sidechains; and amino acids with polar uncharged side chains (i.e., Ser,Thr, Asn, and Gin) can be substituted with other amino acids with polaruncharged side chains.

Treating a subject can comprise delivering an effective amount ordelivering a prophylactic treatment and/or a therapeutic treatment to asubject (e.g., a patient). An “effective amount” is an amount of acompound that can result in a desired physiological change in a subject.Effective amounts may also be administered for research purposes.

A “prophylactic treatment” comprises a treatment administered to asubject who does not display signs or symptoms of a disease orcondition, or a subject who displays only early signs or symptoms of adisease or condition, such that treatment is administered for thepurpose of diminishing, preventing, and/or decreasing the risk offurther developing the disease or condition or of diminishing,preventing, and/or decreasing the risk of developing the disease orcondition. Thus, a prophylactic treatment may function as a preventivetreatment against a disease or condition.

A “therapeutic treatment” comprises a treatment administered to asubject who displays symptoms or signs of a disease or a condition andthe therapeutic treatment is administered to the subject for the purposeof diminishing or eliminating the symptoms or the signs of the diseaseor the condition.

“Therapeutically effective amounts” comprise amounts that provideprophylactic treatment and/or therapeutic treatment. Therapeuticallyeffective amounts need not fully prevent or cure the disease or thecondition but can also provide a partial benefit, such as a delay ofonset or an alleviation or an improvement of at least one symptom of thedisease or the condition.

For administration, effective amounts and therapeutically effectiveamounts (also referred to herein as doses) can be initially estimatedbased on results from in vitro assays and/or animal model studies. Forexample, a dose can be formulated in animal models to achieve acirculating concentration range that includes the IC₅₀ as determined incell culture. Such information can be used to more accurately determineuseful doses in subjects of interest.

The actual dose amount administered to a particular subject can bedetermined by a physician, a veterinarian, or a researcher, taking intoaccount parameters such as, but not limited to, physical andphysiological factors including body weight, severity of condition, typeof disease, previous or concurrent therapeutic interventions, idiopathyof the subject, and/or route of administration.

Doses can range from 1×10⁸ vector genomes per kg (vg/kg) to 1)(10¹⁵vg/kg, from 1×10⁹ vg/kg to 1×10¹⁴ vg/kg, from 1×10¹⁰ vg/kg to 1×10¹³vg/kg, or from 1×10¹¹ vg/kg to 1×10¹² vg/kg. In other non-limitingexamples, a dose can comprise about 1×10⁸ vg/kg, about 1×10⁹ vg/kg,about 1×10¹⁰ vg/kg, about 1×10¹¹ vg/kg, about 1×10¹² vg/kg, about 1×10¹³vg/kg, about 1×10¹⁴ vg/kg, or about 1×10¹⁵ vg/kg. Therapeuticallyeffective amounts can be achieved by administering single or multipledoses during the course of a treatment regimen (i.e., days, weeks,months, etc.).

Pharmaceutically acceptable salts, tautomers, and isomers of thecompounds disclosed herein can also be used. Exemplary salts caninclude, but are not limited to, sulfate, citrate, acetate, oxalate,chloride, bromide, iodide, nitrate, bisulfate, phosphate, acidphosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate,oleate, tannate, pantothenate, bitartrate, ascorbate, succinate,maleate, besylate, gentisinate, fumarate, gluconate, glucaronate,saccharate, formate, benzoate, glutamate, methanesulfonate,ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate(i.e., 1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts.

The formulations described herein can be administered by, withoutlimitation, injection, infusion, perfusion, inhalation, lavage, and/oringestion. Routes of administration can include, but are not limited to,intravenous, intradermal, intraarterial, intraperitoneal, intralesional,intracranial, intraarticular, intraprostatic, intrapleural,intratracheal, intranasal, intravitreal, intravaginal, intrarectal,topically, intratumoral, intramuscular, intravesicular,intrapericardial, intraumbilical, intraocularal, mucosal, oral,subcutaneous, and/or subconjunctival. In other non-limiting examples,administration can be performed by intramuscular injection,intravascular injection, intraperitoneal injection, or any other methodsuitable for delivery of vector to musculature.

In some embodiments, for injection, formulations can be made as aqueoussolutions, such as in buffers including, but not limited to, Hanks'solution, Ringer's solution, and/or physiological saline. The solutionscan contain formulatory agents such as suspending, stabilizing, and/ordispersing agents. Alternatively, the formulation can be in lyophilizedand/or powder form for constitution with a suitable vehicle control(e.g., sterile pyrogen-free water) before use.

Any formulation disclosed herein can advantageously comprise any otherpharmaceutically acceptable carrier or carriers which comprise thosethat do not produce significantly adverse, allergic, or other untowardreactions that may outweigh the benefit of administration, whether forresearch, prophylactic, and/or therapeutic treatments. Exemplarypharmaceutically acceptable carriers and formulations are disclosed inRemington's Pharmaceutical Sciences, 18th Ed., Mack Printing Company,1990, which is incorporated by reference herein for its teachingsregarding the same. Moreover, formulations can be prepared to meetsterility, pyrogenicity, general safety, and purity standards asrequired by the United States FDA's Division of Biological Standards andQuality Control and/or other relevant U.S. and foreign regulatoryagencies.

Exemplary, generally used pharmaceutically acceptable carriers maycomprise, but are not limited to, bulking agents or fillers, solvents orco-solvents, dispersion media, coatings, surfactants, antioxidants(e.g., ascorbic acid, methionine, and vitamin E), preservatives,isotonic agents, absorption delaying agents, salts, stabilizers,buffering agents, chelating agents (e.g., EDTA), gels, binders,disintegration agents, and/or lubricants.

Exemplary buffering agents may comprise, but are not limited to, citratebuffers, succinate buffers, tartrate buffers, fumarate buffers,gluconate buffers, oxalate buffers, lactate buffers, acetate buffers,phosphate buffers, histidine buffers, and/or trimethylamine salts.

Exemplary preservatives may comprise, but are not limited to, phenol,benzyl alcohol, meta-cresol, methylparaben, propyl paraben,octadecyldimethylbenzyl ammonium chloride, benzalkonium halides,hexamethonium chloride, alkyl parabens (such as methyl or propylparaben), catechol, resorcinol, cyclohexanol, and/or 3-pentanol.

Exemplary isotonic agents may comprise polyhydric sugar alcoholscomprising, but not limited to, trihydric or higher sugar alcohols,(e.g., glycerin, erythritol, arabitol, xylitol, sorbitol, and/ormannitol).

Exemplary stabilizers may comprise, but are not limited to, organicsugars, polyhydric sugar alcohols, polyethylene glycol,sulfur-containing reducing agents, amino acids, low molecular weightpolypeptides, proteins, immunoglobulins, hydrophilic polymers, and/orpolysaccharides.

Formulations can also be depot preparations. In some embodiments, suchlong-acting formulations may be administered by, without limitation,implantation (e.g., subcutaneously or intramuscularly) or byintramuscular injection. Thus, for example, compounds can be formulatedwith suitable polymeric and/or hydrophobic materials (e.g., as anemulsion in an acceptable oil) or ion exchange resins, or as sparinglysoluble derivatives (e.g., as a sparingly soluble salt).

Additionally, in various embodiments, compounds can be delivered usingsustained-release systems, such as semipermeable matrices of solidpolymers comprising at least one compound. Various sustained-releasematerials have been established and are well known by those of ordinaryskill in the art. Sustained-release capsules may, depending on theirchemical nature, release the compound following administration for a fewweeks up to over 100 days.

Gene therapy methods can be used for delivering (e.g., at sustainedlevels) specific proteins into patients or subjects. These methods allowpractitioners to introduce DNA coding for a gene of interest directlyinto a patient or subject (in vivo gene therapy) or into cells isolatedfrom a patient, a subject, or a donor (ex vivo gene therapy). Theintroduced DNA then directs the patient's or subject's own cells orgrafted cells to produce the desired protein product. Gene delivery,therefore, can obviate the need for daily injections. Gene therapy mayalso allow practitioners to select specific organs or cellular targets(e.g., muscle, liver, blood cells, brain cells, etc.) for therapy.

DNA may be introduced into a subject's cells in several ways. There aretransfection methods, including chemical methods such as calciumphosphate precipitation and liposome-mediated transfection, and physicalmethods such as electroporation. In general, transfection methods arenot suitable for in vivo gene delivery. There are also methods that userecombinant viruses. Current viral-mediated gene delivery methodsinclude, but are not limited to, retrovirus, adenovirus, herpes virus,pox virus, and adeno-associated virus (AAV) vectors.

One viral system that has been used for gene delivery isadeno-associated virus (AAV). AAV is a parvovirus which belongs to thegenus Dependoparvovirus. AAV has several attractive features not foundin other viruses. First, AAV can infect a wide range of host cells,including non-dividing cells. Second, AAV can infect cells fromdifferent species. Third, AAV has not been associated with any human oranimal disease and does not appear to alter the biological properties ofthe host cell upon integration. Indeed, it is estimated that 80-85% ofthe human population has been exposed to the virus. Finally, AAV isstable at a wide range of physical and chemical conditions which lendsitself to production, storage, and transportation requirements.

The AAV genome is a linear, single-stranded DNA molecule containing 4681nucleotides. The AAV genome generally comprises an internalnon-repeating genome flanked on each end by inverted terminal repeats(ITRs). The ITRs are approximately 145 base pairs (bp) in length. TheITRs have multiple functions, including as origins of DNA replicationand as packaging signals for the viral genome.

The internal non-repeated portion of the genome includes two large openreading frames, known as the AAV replication (rep) and capsid (cap)genes. The rep and cap genes code for viral proteins that allow thevirus to replicate and package the viral genome into a virion. Inparticular, a family of at least four viral proteins are expressed fromthe AAV rep region, Rep78, Rep68, Rep52, and Rep40, named according totheir apparent molecular weight. The AAV cap region encodes at leastthree proteins, VP1, VP2, and VP3.

AAV is a helper-dependent virus; that is, it requires co-infection witha helper virus (e.g., adenovirus, herpesvirus, or vaccinia) in order toform AAV virions. In the absence of co-infection with a helper virus,AAV establishes a latent state in which the viral genome inserts into ahost cell chromosome, but infectious virions are not produced.Subsequent infection by a helper virus “rescues” the integrated genome,allowing it to replicate and package its genome into infectious AAVvirions. While AAV can infect cells from different species, the helpervirus must be of the same species as the host cell. Thus, for example,human AAV will replicate in canine cells co-infected with a canineadenovirus.

“Gene transfer” or “gene delivery” comprises methods or systems forinserting foreign DNA into host cells. Gene transfer can result intransient expression of non-integrated transferred DNA, extrachromosomalreplication, and expression of transferred replicons (e.g., episomes),or integration of transferred genetic material into the genomic DNA ofhost cells.

A “vector” comprises any genetic element, such as, but not limited to, aplasmid, phage, transposon, cosmid, chromosome, artificial chromosome,virus, virion, etc., which is capable of replication when associatedwith the proper control elements and which can transfer gene sequencesbetween cells. Thus, the term includes cloning and expression vehicles,as well as viral vectors.

An “AAV vector” comprises a vector derived from an adeno-associatedvirus serotype, including without limitation, AAV1, AAV2, AAV3, AAV4,AAV5, AAV6, AAV7, AAV8, and AAV9. AAV vectors can have one or more ofthe AAV wild-type genes deleted in whole or part, e.g., the rep and/orcap genes, but retain functional flanking ITR sequences. Functional ITRsequences are necessary for the rescue, replication, and packaging ofthe AAV virion. Thus, an AAV vector is defined herein to include atleast those sequences required in cis for replication and packaging(e.g., functional ITRs) of the virus. The ITRs need not be the wild-typenucleotide sequences, and may be altered, e.g., by the insertion,deletion or substitution of nucleotides, so long as the sequencesprovide for functional rescue, replication and packaging.

A “recombinant AAV vector” or “rAAV vector” comprises an infectious,replication-defective virus composed of an AAV protein shellencapsulating a heterologous nucleotide sequence of interest that isflanked on both sides by AAV ITRs. An rAAV vector is produced in asuitable host cell comprising an AAV vector, AAV helper functions, andaccessory functions. In this manner, the host cell is rendered capableof encoding AAV polypeptides that are required for packaging the AAVvector (containing a recombinant nucleotide sequence of interest) intoinfectious recombinant virion particles for subsequent gene delivery.

A first aspect of the disclosure relates to nucleotide sequencesincluding a micro-dystrophin gene encoding a protein. The nucleotidesequences may also include a regulatory cassette. Additionally, thenucleotide sequences may be isolated and/or purified.

In some embodiments, the protein encoded by the micro-dystrophin genemay include an amino-terminal actin-binding domain, adystroglycan-binding domain, and/or a spectrin-like repeat domain. Thespectrin-like repeat domain may include at least four spectrin-likerepeats or portions of at least four spectrin-like repeats. Two of theat least four spectrin-like repeats may comprise a neuronal nitric oxidesynthase binding domain. Stated another way, the at least fourspectrin-like repeats may include spectrin-like repeats 16 and 17 orportions thereof. In some embodiments, the at least four spectrin-likerepeats may include spectrin-like repeats 1 and 24 or portions thereof.In alternative embodiments, the at least four spectrin-like repeats mayinclude other suitable spectrin-like repeats or portions thereof (e.g.,spectrin-like repeats 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,18, 19, 20, 21, 22, and/or 23).

In certain embodiments, the spectrin-like repeat domain may includefour, five, six, seven, eight, or more spectrin-like repeats or portionsthereof. In certain other embodiments, the protein encoded by themicro-dystrophin gene may include between five spectrin-like repeats andeight spectrin-like repeats (e.g., five, six, seven, or eightspectrin-like repeats). In yet certain other embodiments, thespectrin-like repeat domain may include another suitable number ofspectrin-like repeats or portions thereof.

In some embodiments, the protein encoded by the micro-dystrophin genemay further comprise a hinge domain or a portion thereof. For example,the protein encoded by the micro-dystrophin gene may include at least aportion of a hinge domain selected from at least one of a Hinge 1domain, a Hinge 2 domain, a Hinge 3 domain, a Hinge 4 domain, and/or ahinge-like domain (such as the hinge-like domains encoded by thesequences downstream from spectrin-like repeat 15 (SEQ ID NO:20) andwithin spectrin-like repeat 23 (SEQ ID NO:21)).

In various embodiments, the micro-dystrophin gene may include a portionof the nucleic acid sequence of SEQ ID NO:16. In various otherembodiments, the micro-dystrophin gene may have at least 20%, at least30%, at least 40%, at least 50%, at least 60%, at least 70%, at least75%, at least 76%, at least 77%, at least 78%, at least 79%, at least80%, at least 81%, at least 82%, at least 83%, at least 84%, at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% sequenceidentity to the nucleic acid sequence of SEQ ID NO:16. In yet variousother embodiments, the micro-dystrophin gene may have 100% sequenceidentity to the nucleic acid sequence of SEQ ID NO:16.

In some embodiments, the protein encoded by the micro-dystrophin genemay include a portion of the amino acid sequence of SEQ ID NO:4. In someother embodiments, the protein encoded by the micro-dystrophin gene mayhave at least 20%, at least 30%, at least 40%, at least 50%, at least60%, at least 70%, at least 75%, at least 76%, at least 77%, at least78%, at least 79%, at least 80%, at least 81%, at least 82%, at least83%, at least 84%, at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% sequence identity to the amino acid sequence of SEQID NO:4. In yet some other embodiments, the protein encoded by themicro-dystrophin gene may have 100% sequence identity to the amino acidsequence of SEQ ID NO:4.

In certain embodiments, the micro-dystrophin gene may include a portionof the nucleic acid sequence of SEQ ID NO:18. In certain otherembodiments, the micro-dystrophin gene may have at least 20%, at least30%, at least 40%, at least 50%, at least 60%, at least 70%, at least75%, at least 76%, at least 77%, at least 78%, at least 79%, at least80%, at least 81%, at least 82%, at least 83%, at least 84%, at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% sequenceidentity to the nucleic acid sequence of SEQ ID NO:18. In yet variousother embodiments, the micro-dystrophin gene may have 100% sequenceidentity to the nucleic acid sequence of SEQ ID NO:18.

In various embodiments, the protein encoded by the micro-dystrophin genemay include a portion of the amino acid sequence of SEQ ID NO:5. Invarious other embodiments, the protein encoded by the micro-dystrophingene may have at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 75%, at least 76%, at least 77%, atleast 78%, at least 79%, at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% sequence identity to the amino acid sequenceof SEQ ID NO:5. In yet various other embodiments, the protein encoded bythe micro-dystrophin gene may have 100% sequence identity to the aminoacid sequence of SEQ ID NO:5.

Further, the micro-dystrophin gene may include a portion of one or moreof the nucleic acid sequences of SEQ ID NOs:11-18. In certainembodiments, the protein encoded by the micro-dystrophin gene mayinclude a portion of one or more of the amino acid sequences of SEQ IDNO:3-10. In certain other embodiments, the protein encoded by themicro-dystrophin gene may include a portion of one or more of theproteins depicted in the protein structure diagrams of FIG. 7 (e.g.,μDysH3 and μDys1-μDys16).

In some embodiments, the regulatory cassette may be selected from atleast one of a CK8 promoter, a cardiac troponin T (cTnT) promoter,and/or another suitable regulatory cassette. In certain embodiments, theregulatory cassette may be the CK8 promoter and the CK8 promoter maycomprise a portion of the nucleic acid sequence of SEQ ID NO:19. Incertain other embodiments, the regulatory cassette may be the CK8promoter and the CK8 promoter may have at least at least 20%, at least30%, at least 40%, at least 50%, at least 60%, at least 70%, at least75%, at least 76%, at least 77%, at least 78%, at least 79%, at least80%, at least 81%, at least 82%, at least 83%, at least 84%, at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% sequenceidentity to the nucleic acid sequence of SEQ ID NO:19. In yet certainother embodiments, the regulatory cassette may be the CK8 promoter andthe CK8 promoter may have 100% sequence identity to the nucleic acidsequence of SEQ ID NO:19.

In various embodiments, the regulatory cassette may be the cTnT promoterand the cTnT promoter may comprise a portion of the nucleic acidsequence of SEQ ID NO:1. In various other embodiments, the regulatorycassette may be the cTnT promoter and the cTnT promoter may have atleast at least 20%, at least 30%, at least 40%, at least 50%, at least60%, at least 70%, at least 75%, at least 76%, at least 77%, at least78%, at least 79%, at least 80%, at least 81%, at least 82%, at least83%, at least 84%, at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% sequence identity to the nucleic acid sequence ofSEQ ID NO:1. In yet various other embodiments, the regulatory cassettemay be the cTnT promoter and the cTnT promoter may have 100% sequenceidentity to the nucleic acid sequence of SEQ ID NO:1.

Another aspect of the disclosure relates to pharmaceutical compositionscomprising nucleotide sequences as discussed above. In some embodiments,the pharmaceutical compositions may further include a delivery vehicle.For example, the pharmaceutical compositions may comprise a nucleotidesequence including a regulatory cassette and a micro-dystrophin geneencoding a protein and the pharmaceutical compositions may furthercomprise a delivery vehicle. The nucleotide sequences of thepharmaceutical compositions may be isolated and purified nucleotidesequences.

In various embodiments, the delivery vehicle may comprise anadeno-associated virus (AAV) vector or a recombinant adeno-associatedvirus (rAAV) vector. The AAV vector may be a serotype 6 AAV (AAV6).Likewise, the rAAV vector may be a serotype 6 rAAV (rAAV6). The AAVvector may be a serotype 8 AAV (AAV8). Likewise, the rAAV vector may bea serotype 8 rAAV (rAAV8). The AAV vector may be a serotype 9 AAV(AAV9). Likewise, the rAAV vector may be a serotype 9 rAAV (rAAV9). TherAAV vector may be comprised of AAV2 genomic inverted terminal repeat(ITR) sequences pseudotyped with capsid proteins derived from AAVserotype 6 (rAAV2/6). Other suitable serotypes of the AAV or rAAV arealso within the scope of this disclosure.

In some embodiments, as discussed above, the delivery vehicle mayexpress, or be configured to express, the micro-dystrophin gene. Invarious embodiments, the micro-dystrophin gene may include a portion ofthe nucleic acid sequence of SEQ ID NO:16. In various other embodiments,the micro-dystrophin gene may have at least 20%, at least 30%, at least40%, at least 50%, at least 60%, at least 70%, at least 75%, at least76%, at least 77%, at least 78%, at least 79%, at least 80%, at least81%, at least 82%, at least 83%, at least 84%, at least 85%, at least86%, at least 87%, at least 88%, at least 89%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% sequence identity tothe nucleic acid sequence of SEQ ID NO:16. In yet various otherembodiments, the micro-dystrophin gene may have 100% sequence identityto the nucleic acid sequence of SEQ ID NO:16.

In some embodiments, the protein encoded by the micro-dystrophin genemay include a portion of the amino acid sequence of SEQ ID NO:4. In someother embodiments, the protein encoded by the micro-dystrophin gene mayhave at least 20%, at least 30%, at least 40%, at least 50%, at least60%, at least 70%, at least 75%, at least 76%, at least 77%, at least78%, at least 79%, at least 80%, at least 81%, at least 82%, at least83%, at least 84%, at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% sequence identity to the amino acid sequence of SEQID NO:4. In yet some other embodiments, the protein encoded by themicro-dystrophin gene may have 100% sequence identity to the amino acidsequence of SEQ ID NO:4.

In certain embodiments, the micro-dystrophin gene may include a portionof the nucleic acid sequence of SEQ ID NO:18. In certain otherembodiments, the micro-dystrophin gene may have at least 20%, at least30%, at least 40%, at least 50%, at least 60%, at least 70%, at least75%, at least 76%, at least 77%, at least 78%, at least 79%, at least80%, at least 81%, at least 82%, at least 83%, at least 84%, at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% sequenceidentity to the nucleic acid sequence of SEQ ID NO:18. In yet certainother embodiments, the micro-dystrophin gene may have 100% sequenceidentity to the nucleic acid sequence of SEQ ID NO:18.

In various embodiments, the protein encoded by the micro-dystrophin genemay include a portion of the amino acid sequence of SEQ ID NO:5. Invarious other embodiments, the protein encoded by the micro-dystrophingene may have at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 75%, at least 76%, at least 77%, atleast 78%, at least 79%, at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% sequence identity to the amino acid sequenceof SEQ ID NO:5. In yet various other embodiments, the protein encoded bythe micro-dystrophin gene may have 100% sequence identity to the aminoacid sequence of SEQ ID NO:5.

Also, as discussed above, the regulatory cassette may be selected fromat least one of a CK8 promoter, a cardiac troponin T (cTnT) promoter,and/or another suitable regulatory cassette. In certain embodiments, theregulatory cassette may be the CK8 promoter and the CK8 promoter maycomprise a portion of the nucleic acid sequence of SEQ ID NO:19. Incertain other embodiments, the regulatory cassette may be the CK8promoter and the CK8 promoter may have at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 75%, atleast 76%, at least 77%, at least 78%, at least 79%, at least 80%, atleast 81%, at least 82%, at least 83%, at least 84%, at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% sequence identityto the nucleic acid sequence of SEQ ID NO:19. In yet certain otherembodiments, the regulatory cassette may be the CK8 promoter and the CK8promoter may have 100% sequence identity to the nucleic acid sequence ofSEQ ID NO:19.

In various embodiments, the regulatory cassette may be the cTnT promoterand the cTnT promoter may comprise a portion of the nucleic acidsequence of SEQ ID NO:1. In various other embodiments, the regulatorycassette may be the cTnT promoter and the cTnT promoter may have atleast at least 20%, at least 30%, at least 40%, at least 50%, at least60%, at least 70%, at least 75%, at least 76%, at least 77%, at least78%, at least 79%, at least 80%, at least 81%, at least 82%, at least83%, at least 84%, at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% sequence identity to the nucleic acid sequence ofSEQ ID NO:1. In yet various other embodiments, the regulatory cassettemay be the cTnT promoter and the cTnT promoter may have 100% sequenceidentity to the nucleic acid sequence of SEQ ID NO:1.

In some embodiments, the pharmaceutical composition may be configured toreduce a pathological effect or symptom of a muscular dystrophy. Themuscular dystrophy may be selected from at least one of myotonicmuscular dystrophy, Duchenne muscular dystrophy, Becker musculardystrophy, limb-girdle muscular dystrophy, facioscapulohumeral musculardystrophy, congenital muscular dystrophy, oculopharyngeal musculardystrophy, distal muscular dystrophy, Emery-Dreifuss muscular dystrophy,and/or another suitable muscular dystrophy. In some other embodiments,the pharmaceutical composition may be configured to reduce apathological effect or symptom of a muscular dystrophy selected from atleast one of Duchenne muscular dystrophy and/or Becker musculardystrophy. In certain embodiments, the pharmaceutical composition may beconfigured to reduce a pathological effect or symptom of at least one ofsarcopenia, heart disease, and/or cachexia.

Another aspect of the disclosure relates to methods for treating asubject having muscular dystrophy, sarcopenia, heart disease, and/orcachexia. The methods may comprise administering to the subject apharmaceutical composition comprising a micro-dystrophin gene coupled toa regulatory cassette. The methods may comprise administering to thesubject a therapeutically effective amount of the pharmaceuticalcomposition. Furthermore, the micro-dystrophin gene may be operablycoupled to the regulatory cassette.

In some embodiments, the method may comprise administering to thesubject a pharmaceutical composition wherein the pharmaceuticalcomposition further comprises an AAV vector, an rAAV vector, and/oranother suitable delivery vehicle. The delivery vehicle may express, orbe configured to express, the micro-dystrophin gene in the subject.

As discussed above, in various embodiments, the micro-dystrophin genemay include a portion of the nucleic acid sequence of SEQ ID NO:16. Invarious other embodiments, the micro-dystrophin gene may have at least20%, at least 30%, at least 40%, at least 50%, at least 60%, at least70%, at least 75%, at least 76%, at least 77%, at least 78%, at least79%, at least 80%, at least 81%, at least 82%, at least 83%, at least84%, at least 85%, at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% sequence identity to the nucleic acid sequence of SEQ ID NO:16. Inyet various other embodiments, the micro-dystrophin gene may have 100%sequence identity to the nucleic acid sequence of SEQ ID NO:16.

In some embodiments, the protein encoded by the micro-dystrophin genemay include a portion of the amino acid sequence of SEQ ID NO:4. In someother embodiments, the protein encoded by the micro-dystrophin gene mayhave at least 20%, at least 30%, at least 40%, at least 50%, at least60%, at least 70%, at least 75%, at least 76%, at least 77%, at least78%, at least 79%, at least 80%, at least 81%, at least 82%, at least83%, at least 84%, at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% sequence identity to the amino acid sequence of SEQID NO:4. In yet some other embodiments, the protein encoded by themicro-dystrophin gene may have 100% sequence identity to the amino acidsequence of SEQ ID NO:4.

In certain embodiments, the micro-dystrophin gene may include a portionof the nucleic acid sequence of SEQ ID NO:18. In certain otherembodiments, the micro-dystrophin gene may have at least 20%, at least30%, at least 40%, at least 50%, at least 60%, at least 70%, at least75%, at least 76%, at least 77%, at least 78%, at least 79%, at least80%, at least 81%, at least 82%, at least 83%, at least 84%, at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% sequenceidentity to the nucleic acid sequence of SEQ ID NO:18. In yet certainother embodiments, the micro-dystrophin gene may have 100% sequenceidentity to the nucleic acid sequence of SEQ ID NO:18.

In various embodiments, the protein encoded by the micro-dystrophin genemay include a portion of the amino acid sequence of SEQ ID NO:5. Invarious other embodiments, the protein encoded by the micro-dystrophingene may have at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 75%, at least 76%, at least 77%, atleast 78%, at least 79%, at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% sequence identity to the amino acid sequenceof SEQ ID NO:5. In yet various other embodiments, the protein encoded bythe micro-dystrophin gene may have 100% sequence identity to the aminoacid sequence of SEQ ID NO:5.

In some embodiments, the regulatory cassette may express, or beconfigured to express, the micro-dystrophin gene such that a level ofexpression of the micro-dystrophin gene is at least 100-fold higher instriated muscle cells than the level of expression of themicro-dystrophin gene in non-muscle cells. For example, the level ofexpression of the micro-dystrophin gene may be at least 100-fold higherin the striated muscle cells of the subject than in lung cells of thesubject. In some other embodiments, the regulatory cassette may express,or be configured to express, the micro-dystrophin gene such that a levelof expression of the micro-dystrophin gene is between at least 50-foldhigher and 150-fold higher, between at least 75-fold higher and 125-foldhigher, or between at least 90-fold higher and 110-fold higher instriated muscle cells than the level of expression of themicro-dystrophin gene in non-muscle cells.

As discussed above, the regulatory cassette may be selected from atleast one of a CK8 promoter, a cardiac troponin T (cTnT) promoter,and/or another suitable regulatory cassette. In certain embodiments, theregulatory cassette may be the CK8 promoter and the CK8 promoter maycomprise a portion of the nucleic acid sequence of SEQ ID NO:19. Incertain other embodiments, the regulatory cassette may be the CK8promoter and the CK8 promoter may have at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 75%, atleast 76%, at least 77%, at least 78%, at least 79%, at least 80%, atleast 81%, at least 82%, at least 83%, at least 84%, at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% sequence identityto the nucleic acid sequence of SEQ ID NO:19. In yet certain otherembodiments, the regulatory cassette may be the CK8 promoter and the CK8promoter may have 100% sequence identity to the nucleic acid sequence ofSEQ ID NO:19.

In various embodiments, the regulatory cassette may be the cTnT promoterand the cTnT promoter may comprise a portion of the nucleic acidsequence of SEQ ID NO:1. In various other embodiments, the regulatorycassette may be the cTnT promoter and the cTnT promoter may have atleast at least 20%, at least 30%, at least 40%, at least 50%, at least60%, at least 70%, at least 75%, at least 76%, at least 77%, at least78%, at least 79%, at least 80%, at least 81%, at least 82%, at least83%, at least 84%, at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% sequence identity to the nucleic acid sequence ofSEQ ID NO:1. In yet various other embodiments, the regulatory cassettemay be the cTnT promoter and the cTnT promoter may have 100% sequenceidentity to the nucleic acid sequence of SEQ ID NO:1.

In some embodiments, the micro-dystrophin gene may express, or beconfigured to express, a micro-dystrophin protein in one or more musclesof the subject such that contractility of the one or more muscles isenhanced or increased. In certain embodiments, the micro-dystrophin genemay express, or be configured to express, a micro-dystrophin protein inone or more skeletal muscles of the subject such that a specific-forcegenerating capacity of at least one of the one or more skeletal musclesis enhanced or increased to within at least 10%, at least 20%, at least30%, or at least 40% of a normal specific-force generating capacity. Incertain other embodiments, the micro-dystrophin gene may express, or beconfigured to express, a micro-dystrophin protein in one or more cardiacmuscles of the subject such that a baseline end-diastolic volume defectis restored to within at least 10%, at least 20%, at least 30%, or atleast 40% of a normal end-diastolic volume. In various embodiments, themicro-dystrophin gene may express, or be configured to express, amicro-dystrophin protein such that localization of the neuronal nitricoxide synthase to the dystrophin-glycoprotein complex is enhanced orincreased in the subject.

In some embodiments, as discussed above, the methods may comprisetreating a subject having at least one of myotonic muscular dystrophy,Duchenne muscular dystrophy, Becker muscular dystrophy, limb-girdlemuscular dystrophy, facioscapulohumeral muscular dystrophy, congenitalmuscular dystrophy, oculopharyngeal muscular dystrophy, distal musculardystrophy, Emery-Dreifuss muscular dystrophy, and/or another suitablemuscular dystrophy. In some other embodiments, the methods may comprisetreating a subject having at least one of Duchenne muscular dystrophyand/or Becker muscular dystrophy.

In certain embodiments, the pharmaceutical composition may reduce, or beconfigured to reduce, a pathological effect or symptom of the musculardystrophy, sarcopenia, heart disease, and/or cachexia. The pathologicaleffect or symptom of the muscular dystrophy may be selected from atleast one of muscle pain, muscle weakness, muscle fatigue, muscleatrophy, fibrosis, inflammation, increase in average myofiber diameterin skeletal muscle, cardiomyopathy, reduced 6-minute walk test time,loss of ambulation, cardiac pump failure, and/or one or more othersuitable pathological effects or symptoms. The pathological effect orsymptom of sarcopenia may be selected from at least one of musclewasting and/or muscle weakness. The pathological effect or symptom ofheart disease may be selected from at least one of cardiomyopathy,reduced hemodynamics, and/or arrhythmia. The pathological effect orsymptom of cachexia may be selected from at least one of muscle wastingand/or muscle weakness.

The methods of treating a subject having muscular dystrophy may furthercomprise identifying a subject having muscular dystrophy. Similarly, themethods of treating a subject having sarcopenia, heart disease, and/orcachexia may further comprise identifying a subject having sarcopenia,heart disease, and/or cachexia, respectively. In some embodiments, thesubject may be a mammal. In certain embodiments, the subject may be ahuman.

Another aspect of the disclosure relates to methods for prophylacticallytreating a subject at risk of developing muscular dystrophy, sarcopenia,heart disease, and/or cachexia. The methods may comprise administeringto the subject a pharmaceutical composition as described above inreference to the methods of treating a subject having a musculardystrophy, sarcopenia, heart disease, and/or cachexia.

In some embodiments, the methods may comprise treating a subject at riskof developing at least one of myotonic muscular dystrophy, Duchennemuscular dystrophy, Becker muscular dystrophy, limb-girdle musculardystrophy, facioscapulohumeral muscular dystrophy, congenital musculardystrophy, oculopharyngeal muscular dystrophy, distal musculardystrophy, Emery-Dreifuss muscular dystrophy, and/or another suitablemuscular dystrophy. In some other embodiments, the methods may comprisetreating a subject at risk of developing at least one of Duchennemuscular dystrophy and/or Becker muscular dystrophy.

In certain embodiments, the pharmaceutical composition may reduce, or beconfigured to reduce, a risk of developing a pathological effect orsymptom of a muscular dystrophy, sarcopenia, heart disease, and/orcachexia. The methods of treating a subject at risk of developingmuscular dystrophy, sarcopenia, heart disease, and/or cachexia mayfurther comprise identifying a subject at risk of developing musculardystrophy, sarcopenia, heart disease, and/or cachexia, respectively. Insome embodiments, the subject may be a mammal. In certain embodiments,the subject may be a human.

Another aspect of the disclosure relates to regulatory cassettesincluding enhancers and/or promoters that enhance and/or targetexpression of a pharmaceutical composition (e.g., a micro-dystrophingene). In some embodiments, the enhancers or promoters for enhancingand/or targeting expression of a pharmaceutical composition may includeat least a portion of a gene, a peptide, a polypeptide, and/or aregulatory RNA. Targeting expression of the pharmaceutical compositionmay include expression the pharmaceutical composition in a specific celltype, tissue, and/or organ of a subject. For example, cTnT455 (SEQ IDNO:1) may be used for cardiac-specific expression.

In certain embodiments, the enhancers or promoters may express, or beconfigured to express, a pharmaceutical composition comprising apeptide. In various embodiments, the enhancers or promoters may express,or be configured to express, the peptide in developing, injured, and/ordiseased muscle (i.e., muscle that may be undergoing regeneration). Thehum-cTnT455 RC (SEQ ID NO:1) may not be transcriptionally active insteady state mature skeletal muscle.

As discussed above, the enhancers and/or promoters may be operativelylinked to a pharmaceutical composition, i.e., for enhancing expressionand/or targeting of the pharmaceutical composition. Additionally, thepharmaceutical composition may be operatively linked to one or enhancersand/or promoters. In some embodiments, expression of the pharmaceuticalcompositions disclosed herein may assist in regenerating cardiac muscle.For example, the hum-cTnT455 RC (SEQ ID NO:1) may enhance or target thetransient expression of the pharmaceutical composition in wounded and/orregenerating cardiac muscle. In some embodiments, expression of thepharmaceutical compositions disclosed herein may assist in preventingloss of cardiac muscle and/or of cardiomyocytes. In certain embodiments,expression of the pharmaceutical compositions disclosed herein mayassist in regenerating skeletal muscle. In various embodiments,expression of the pharmaceutical compositions disclosed herein mayassist in preventing necrosis and/or wasting of skeletal muscle.

Another aspect of the disclosure relates to nucleotide sequencescomprising a micro-dystrophin gene, wherein the micro-dystrophin genemay encode a protein comprising at least two spectrin-like repeats thatare directly coupled to each other. In some embodiments, the at leasttwo spectrin-like repeats that are directly coupled to each other may beselected from at least one of a spectrin-like repeat 1 (SR1) directlycoupled to a spectrin-like repeat 2 (SR2), an SR2 directly coupled to aspectrin-like repeat 3 (SR3), an SR1 directly coupled to a spectrin-likerepeat 16 (SR16), a spectrin-like repeat 17 (SR17) directly coupled to aspectrin-like repeat 23 (SR23), an SR17 directly coupled to aspectrin-like repeat 24 (SR24), and/or an SR23 directly coupled to anSR24. The micro-dystrophin gene may also encode a protein comprising anamino-terminal actin-binding domain and/or a β-dystroglycan bindingdomain.

Another aspect of the disclosure relates to nucleotide sequencescomprising a micro-dystrophin gene, wherein the micro-dystrophin genemay encode a protein comprising, in order, a Hinge 1 domain (H1), anSR1, an SR16, an SR17, an SR24, and/or a Hinge 4 domain (H4). In someembodiments, the H1 may be directly coupled to the SR1. In variousembodiments, the SR 1 may be directly coupled to the SR16. In certainembodiments, the SR16 may be directly coupled to the SR17. In someembodiments, the SR17 may be directly coupled to the SR24. In variousembodiments, the SR24 may be directly coupled to the H4.

In some embodiments, the protein encoded by the micro-dystrophin genemay further comprise, in order, an SR2 and an SR3, wherein the SR2 andthe SR3 may be disposed between the SR1 and the SR16. Furthermore, theSR1 may be directly coupled to the SR2 and the SR2 may be furthercoupled to the SR3.

Another aspect of the disclosure relates to nucleotide sequencescomprising a micro-dystrophin gene encoding a protein, wherein themicro-dystrophin gene may encode a protein comprising, in order, a H1,an SR1, an SR16, an SR17, an SR 23, an SR24, and/or a H4. In someembodiments, the H1 may be directly coupled to the SR1, the SR1 may bedirectly coupled to the SR 16, the SR 16 may be directly coupled to theSR 17, the SR 17 may be directly coupled to the SR 23, the SR 23 may bedirectly coupled to the SR 24, and/or the SR 24 may be directly coupledto the H4.

Another aspect of the disclosure relates to pharmaceutical compositionsthat may comprise a micro-dystrophin gene comprising the nucleic acidsequence of SEQ ID NO:16 and an adeno-associated virus (AAV) vector or arecombinant adeno-associated virus (rAAV) vector. In some embodiments, aserotype of the AAV vector or the rAAV vector may selected from at leastone of serotype 6, serotype 8, serotype 9, or another suitable serotype.

Another aspect of the disclosure relates to pharmaceutical compositionthat may comprise a micro-dystrophin gene encoding a protein, whereinthe protein may comprise the amino acid sequence of SEQ ID NO:4 and anAAV vector or an rAAV vector. In certain embodiments, a serotype of theAAV vector or the rAAV vector may be selected from at least one ofserotype 6, serotype 8, serotype 9, or another suitable serotype.

Another aspect of the disclosure relates to pharmaceutical compositionsthat may comprise a micro-dystrophin gene comprising the nucleic acidsequence of SEQ ID NO:18 and an AAV vector or an rAAV vector. In variousembodiments, a serotype of the AAV vector or the rAAV vector is selectedfrom at least one of serotype 6, serotype 8, serotype 9, or anothersuitable serotype.

Another aspect of the disclosure relates to pharmaceutical compositionsthat may comprise a micro-dystrophin gene encoding a protein, whereinthe protein may comprise the amino acid sequence of SEQ ID NO:5 and anAAV vector or an rAAV vector. In some embodiments, a serotype of the AAVvector or the rAAV vector is selected from at least one of serotype 6,serotype 8, serotype 9, or another suitable serotype.

Another aspect of the disclosure relates to pharmaceutical compositionsfor use in the treatment or prophylactic treatment of musculardystrophy, sarcopenia, heart failure, and/or cachexia. In someembodiments, the pharmaceutical compositions may comprise amicro-dystrophin gene. In certain embodiments, the micro-dystrophin genemay comprise the nucleic acid sequence of SEQ ID NO:16 or SEQ ID NO:18and an AAV vector or an rAAV vector. In various embodiments, a serotypeof the AAV vector or the rAAV vector may be selected from at least oneof serotype 6, serotype 8, serotype 9, or another suitable serotype.

Another aspect of the disclosure relates to pharmaceutical compositionsfor the treatment or prophylactic treatment of muscular dystrophysarcopenia, heart failure, and/or cachexia. In some embodiments, thepharmaceutical compositions may comprise a micro-dystrophin gene. Incertain embodiments, the micro-dystrophin gene may comprise the nucleicacid sequence of SEQ ID NO:16 or SEQ ID NO:18 and an AAV vector or anrAAV vector. In various embodiments, a serotype of the AAV vector or therAAV vector may be selected from at least one of serotype 6, serotype 8,serotype 9, or another suitable serotype.

EXAMPLES

The following examples are illustrative of disclosed methods andcompositions. In light of this disclosure, those of skill in the artwill recognize that variations of these examples and other examples ofthe disclosed methods and compositions would be possible without undueexperimentation.

Example 1—Development of Micro-Dystrophins

To develop micro-dystrophins with improved performance a variety ofstructural modifications of the dystrophin central rod domain, whichaccounts for approximately 80% of the coding region, were assessed.Novel constructs were generated that comprise unique combinations ofbetween four and six of the 24 spectrin-like repeats (SRs) present inthe full-length protein as well as the presence or absence of internalhinge domains. These novel micro-dystrophins were evaluated byrAAV-mediated delivery to dystrophic mdx mice followed bypathophysiologic analysis of skeletal muscles after three and sixmonths.

Several versions of μDys clones were designed with a focus on increasingfunctional activity while allowing more complete restoration of thedystrophin glycoprotein complex (DGC). The designed μDys clones werecompared with a previously characterized ΔH2-R23+H3/ΔCT clone, μDysH3,which can be highly functional in striated muscles of mdx mice (seeBanks, G. B., et al., PLoS Genetics 6, e1000958, (2010)). The design ofthese constructs focused, at least in part, on the central rod domain inefforts to improve the contractility of muscles expressing theconstructs and to restore neuronal nitric oxide synthase (nNOS)localization to the DGC (see Lai, Y., et al., The Journal of ClinicalInvestigation 119, 624-635, (2009) and Lai, Y., et al., Proceedings ofthe National Academy of Sciences of the United States of America 110,525-530, (2013)). Also tested, were the functional capacity and theability to deliver larger constructs carrying 4, 5, or 6 SRs. To allowstable packaging of these larger μDys clones, a small gene regulatorycassette (RC) modified from the muscle creatine kinase gene wasincorporated. This CK8 RC can display strong, muscle-restrictedexpression, yet this CK8 RC is less than 500 bps in size (see Goncalves,M. A., et al., Molecular Therapy: The Journal of the American Society ofGene Therapy 19, 1331-1341, (2011) and Martari, M., et al., Human GeneTherapy 20, 759-766, (2009)).

Example 2—Design of Micro-Dystrophin Clones

Seven novel micro-dystrophin (μDys) clones were designed to testvariations of the rod domain structure. Each of the sevenmicro-dystrophin clones retained coding sequences for the N-terminalactin-binding domain (N-ABD) and the dystroglycan-binding domain (DgBD), however, each μDys clone incorporated novel combinations of SR andhinge domains, with a goal of generating μDys clones with improvedfunctional properties that may be delivered and expressed from an rAAVvector. Each of the μDys clones were also tested in a mouse model forDuchenne muscular dystrophy (DMD), as described below. The SEQ ID NOs ofthe amino acid sequences and nucleic acid sequences of μDysH3 and theseseven novel μDys constructs are listed in Table 1.

TABLE 1 Micro-dystrophin Construct Sequences Micro-dystrophin Amino AcidNucleic Acid Construct Sequence Sequence μDysH3 SEQ ID NO: 3 SEQ ID NO:11 μDys1 SEQ ID NO: 6 SEQ ID NO: 12 μDys2 SEQ ID NO: 7 SEQ ID NO: 13μDys3 SEQ ID NO: 8 SEQ ID NO: 14 μDys4 SEQ ID NO: 9 SEQ ID NO: 15 μDys5SEQ ID NO: 4 SEQ ID NO: 16 μDys6 SEQ ID NO: 10 SEQ ID NO: 17 μDys7 SEQID NO: 5 SEQ ID NO: 18

Previous studies suggest that the choice of hinge domains within a μDysclone can impact the function of the protein (see Banks, G. B., et al.,PLoS Genetics 6, e1000958, (2010)). It was assessed whether alternativeand/or shorter hinge domains could be substituted for the Hinge 3domain, which was used in the μDys clone, μDysH3 (see id.). It has beenindicated that inclusion of SRs 16 and 17 can improve the function ofsome μDys clones (e.g., by recruiting nNOS to the DGC). Accordingly, SRs16 and 17 were also tested in the context of various hinge domains andother SRs. Creation of novel junctions was also minimized (i.e.,junctions wherein domains not normally adjacent to one another in thefull-length protein are brought together). Additionally, the effect ofthe inclusion of combinations of either 5 or 6 SRs on μDys-clonefunction was also assessed. The structure of the seven novel μDysclones, in comparison to the μDysH3 clone and the full-length protein,are illustrated in FIG. 1A.

Two regions in the dystrophin were tested for their ability tosubstitute for Hinge 3. The hinge regions of the rod domain are prolinerich and lack alpha-helical signature motifs that compose thetriple-helical coiled-coil of a spectrin-like repeat (see Winder, S. J.,et al., FEBS Letters 369, 27-33 (1995)). SR23 contains a proline-richlinker between alpha-helices b and c (see, e.g., FIG. 21). It wasassessed if this sequence (with alpha-helix c of SR23) could be used asa hinge domain either by itself (μDys1), adjoining SR16-17 (μDys2), ortogether with H3 (μDys4). One additional construct replaced Hinge 3 withthe entire SR23 (μDys5; see FIG. 1A). A second hinge-like region (SEQ IDNO:20) composed of a 20 amino acid insertion previously noted to belocated between SR15 and SR16 was also tested (μDys6) (see Winder, S.J., et al., FEBS Letters 369, 27-33 (1995)). Additional constructs weredesigned to test various combinations of the SR domains in the contextof these hinges. It has been suggested that the context of SR domainscan be important for their function, as such, it was tested whether ahybrid SR, composed of the first half of SR20 and the final half ofSR24, would improve μDys function (μDys3). This hybrid SR merges theportion of SR20 normally adjacent to Hinge 3 with the portion of SR24that merges into Hinge 4 (see FIG. 1A). Similar considerationsinfluenced the design of the μDys6 construct noted above, where thenovel hinge located between SR15 and SR16 was used in its normal contextadjacent to the nNOS location region in SR16-17. This latter constructwas also compared directly with a similar construct but which used Hinge3 instead of the short hinge-like region from between SR15 and SR16 (seeFIG. 1B). It was also noted that μDys clones 5-7, which incorporateeither 5 or 6 SR domains, potentially increase the overall function ofthe protein (see Harper, S. Q. et al., Nature Medicine 8, 253-261,(2002)).

Example 3—Functionality of Partial Spectrin-Like Repeats can beDependent on the Rod Domain Composition

An initial functional screen of μDys clones 1-7 was made in comparisonto the μDysH3 clone by generating rAAV6 vectors regulated by the CMVpromoter. A dose of 5×10¹⁰ vector genomes (vg) was intramuscularlyinjected into one tibialis anterior (TA) muscle of 5-6 week olddystrophic mdx^(4cv) male mice (see Chapman, V. M., et al., Proceedingsof the National Academy of Sciences of the United States of America 86,1292-1296 (1989)), with the contralateral muscle serving as an internalnegative control (N=4-5 mice per construct).

Dystrophin expression and central nucleation, a hallmark ofdegeneration/regeneration, was measured at 4 weeks and 12 weekspost-injection (see FIGS. 1C and 1D) to determine how well eachconstruct was expressed, whether expression persisted, and whether theconstructs were able to prevent or reduce ongoing myofiber necrosis. Allconstructs generated μDys proteins of the predicted sizes, as shown byWestern blot analysis (see FIG. 1B). At this age and vector dose perinjected TA muscle, all treated mdx^(4cv) cohorts had significantlyfewer dystrophin-positive (Dys+) myofibers compared to wild type C57BL/6mice (P<0.001), yet differences of functionality were observed among themicro-dystrophins. Constructs μDys3 and μDys4 performed less well thanμDysH3, as evidenced by a reduction in dystrophin-positive myofibersbetween 4 and 12 weeks post-injection. Constructs μDys-1, 2, and 5exhibited more dystrophin-positive myofibers than μDysH3 by 12 weekspost-injection (see FIG. 1D). An initial screen of μDys6 and μDys7 wasmade against μDysH3 driven by the CK8 promoter. Both the new constructsgenerated comparable levels of transduced (Dys+) and centrally nucleated(CNF+) myofibers by 12 weeks post-injection relative to μDysH3 (see FIG.1D). Myofibers exhibiting both dystrophin expression and centralnucleation were quantified at both time points (see FIGS. 1C and 1D).Levels of Dys+ and CNF+myofibers decreased from 4 to 12 weekspost-injection in the treated cohorts, yet remained higher than in wildtype muscles. Whether this was the result of poor functionality orsub-optimal dose of a micro-dystrophin construct remained uncertain withthe initial screen alone, which prompted a systemic administration forfurther evaluation.

Example 4—Novel μDys Constructs Attenuate Pathology in Respiratory andHind Limb Skeletal Muscles

The μDys-1, 2, 3, 4, 5, and μDysH3 vectors were re-cloned to replace theCMV with the smaller and muscle-specific CK8 promoter, enabling a directcomparison with the larger six SR-containing constructs (μDys6 and 7).For systemic treatment, a bolus of 10¹³ vg was delivered to 14-day oldmdx^(4cv) male mice via retro-orbital injection. Treated mice wereassessed at either 3 or 6 months post-injection, along with age matcheduntreated and wild type controls. This experiment was designed tomonitor expression of the μDys constructs and assess the relative extentto which they may halt dystrophic pathophysiology. Persistence of μDysexpression was measured by immunofluorescence staining of gastrocnemiusmuscle and diaphragm muscle cryosections. The recruitment of DGCmembers, β-dystroglycan and nNOS (for applicable constructs), to thesarcolemma was also verified (see FIG. 2).

At three months post-injection, all treated groups had greater than 60%expression of dystrophin at the sarcolemma in both the gastrocnemius anddiaphragm myofibers. The percentage of dystrophin-positive myofibersthat were centrally nucleated was not significantly different from wildtype controls (see FIGS. 3A and 3C). At this time point, μDys2 wasobserved to be expressed at significantly lower levels compared withμDys5 in the gastrocnemius and the diaphragm (see FIGS. 3A and 3C). TheμDys2 treated mice also had significantly fewer transduced myofibers inthe diaphragm compared to μDys1, μDys5, and μDysH3 injected animals (seeFIG. 3C). Conversely, μDys5 injected mice displayed significantly highernumbers of transduced myofibers in the gastrocnemius compared with allother treated groups (see FIG. 3A). Morphological analysis of the samemuscles demonstrated that all treated groups had significantly reducedpercentages of centrally nucleated myofibers. In the diaphragm, therewere no significant differences in the percentages of centrallynucleated myofibers between the wild type and treated groups. However,μDys2 and μDysH3 injected mice displayed significantly higher levels ofcentral nucleation (19% and 20%, respectively; P<0.001) than wild type(0%) in the gastrocnemius.

The absence of a functional dystrophin can impair assembly of the DGC.This can result in a loss of mechanical force transmission as well asincreased susceptibility to contraction-induced injury (see Emery, A. E.H. and Muntoni, F., Duchenne Muscular Dystrophy, Third Edition (OxfordUniversity Press, 2003) and Ozawa, E. in Myology (ed. Franzini-ArmstrongC Engel A) 455-470 (McGraw-Hill, 2004)). Expression of some rAAV-μDysvectors has demonstrated an ability to increase specific forcegeneration and resistance to contraction-induced injury in dystrophicanimal models (see Seto, J. T., et al., Current Gene Therapy 12, 139-151(2012)). It was assessed which novel μDys constructs could improve thesemetrics at three months post-injection (see FIGS. 3C and 3D).

Gastrocnemius muscles and diaphragm muscle strips were prepared for insitu and in vitro measurement of mechanical properties, respectively.The specific force generation in the gastrocnemius muscle increased inall treated groups compared to untreated dystrophic controls (see FIG.3B). Only μDys1 and μDys2 injected mice displayed increased specificforce in diaphragm muscle strips (156 kN/m² and 110 kN/m², respectively,compared to 98 kN/m² in untreated mice) (see FIG. 3D). Additionally,expression of all the novel μDys constructs increased resistance tocontraction-induced injury, yet there were no significant differences incomparison to each other. The dystrophic pathology appeared halted bythree months post-injection, yet the physiological performance was notsignificantly improved. Longer time points may be used to further assessthe functionality of the μDys constructs.

Example 5—Long-Term Expression Exposes Functional Discrepancies of μDysConstructs

By six months post-treatment, most treated groups exhibited reducedexpression of dystrophin and a concomitant increase in the percentage ofmyofibers displaying central nucleation, albeit to varying degrees,compared to analysis at three months post-treatment. However, thepercentage of myofibers exhibiting both dystrophin expression andcentral nucleation was not significantly different from wild typecontrols (see FIGS. 4A and 4C). The μDys1, -5, -6, -7 and -H3 injectedmice displayed ≥60% dystrophin positive myofibers in the gastrocnemiusand ≥74% in the diaphragm at 6 months. Transduction levels of μDys2decreased approximately 2-fold in the gastrocnemius over the course ofthree months (from 63% to 31% positive myofibers), and decreased 20% inthe diaphragm, making its performance the worst of the constructs tested(P<0.001; see FIGS. 4A and 4C). The degree of degeneration/regenerationhad increased in both muscles for all treated cohorts, with theexception of two tested constructs. Central nucleation for μDys1remained at 3% in the diaphragm, and μDysH3 decreased from 20% to 8% inthe gastrocnemius (see FIGS. 4A and 4C).

Despite the morphological trend observed with the six monthpost-treatment data, the specific force generation was still higher thanin muscles from untreated controls. Injection of one construct, μDys5,led to force generation levels close to those in wild type mice in boththe gastrocnemius (225 versus 226 kN/m²; see FIG. 4B) and the diaphragm(148 versus 160 kN/m²; see FIG. 4D). Based on previous studies withmini-dystrophins containing six to eight SRs, it was predicted that theμDys constructs containing six SRs would generate the most specificforce as well as provide the greatest protection fromcontraction-induced injury (see Harper, S. Q., et al., Nature Medicine8, 253-261, (2002)). However, specific force generation in thegastrocnemius muscles of μDys6 and μDys7 treated mice was significantlyhigher than untreated controls (P<0.01 and P<0.0001, respectively), butwere not the highest (see FIGS. 4B and 4D). Instead, μDys5 injected micedisplayed the highest levels of specific force generation. The largerconstructs were also not necessarily the best at protecting fromcontraction-induced injury. For example, μDys6 injected mice had thelargest force deficit while μDys7 provided the highest protection fromcontraction-induced injury in the gastrocnemius (see FIG. 5A). However,resistance to contraction-induced injury in diaphragm muscle strips wasthe highest in mice expressing μDys6 and μDys7 as well as μDys5 andμDysH3 (see FIG. 5B). The contrasting results among μDys6 and μDys7between muscle groups and the significant difference in force deficitswithin the gastrocnemius (P<0.0001) suggest that the performance of aparticular μDys construct may be influenced by the regulatory expressioncassette and the muscle assessed (see Harper, S. Q., et al., Naturemedicine 8, 253-261, (2002) and Salva, M. Z., et al., Molecular Therapy:The Journal of the American Society of Gene Therapy 15, 320-329,(2007)). This point was also exemplified with μDys2 treatment, where thesusceptibility to contraction-induced injury was reduced in thegastrocnemius but unexpectedly exacerbated in the diaphragm, relative tountreated controls (see FIGS. 5A and 5B).

Example 6—Gene Delivery Via rAAV6+Cardiac-Specific Promoter

Vectors for WT and L48Q cTnC (rAAV6-WT cTnC and rAAV6-L48Q cTnC,respectively) were produced. Plasmids can be produced with rAAV genomescontaining a cardiac specific promoter (cTnT455) and a C-terminal c-Myctag. cTnC variant transgene expression cassettes (with an mCherryfluorescent reporter) can be co-transfected into HEK293 cells with apackaging/helper plasmid pDGM6 by CaPO₄ precipitation methodology.Vectors can be collected from culture, freeze-thawed, and thesupernatant can be collected. Affinity purification can use a HITRAP™heparin column (GE HEALTHCARE LIFE SCIENCES™, Piscataway, N.J.). Thevirus can be concentrated on a sucrose gradient (40%), spun at 27,000rpm (18 hours, 4° C.), and resolubilized in Hanks' balanced solution.Vector genomes can be determined relative to plasmid standards using aSV40 polyadenylation region oligonucleotide ³²P end-labeled probe withSouthern blot hybridization and confirmed by qPCR.

FIG. 8 depicts the first use of the rAAV6-L48Q cTnC systemicallyinjected (intraocular) into 3 mice each at low (L; 0.6×10¹²) and high(H; 1.2×10¹²) viral particle dose. Echocardiography indicated about a20% increase in left ventricular (LV) ejection fraction compared withuninjected (UN) controls two weeks after injection, and a 30-40%increase at 3 weeks. Systemic injections with control adenoviral vectorshave not altered LV function. Myofibrils from one rAAV6-L48Q cTnC-myctransfected mouse (and uninjected control) were separated by SDS-PAGEand Western blots were probed with anti-cTnC. The presence of myc-tagcaused slower migration of cTnC (see FIG. 9), and the ratio of cTnC-mycto native cTnC was densitometrically determined to be about 40% (similarto that seen with adenovirus and transgenic animals, see below).

Example 7—Acute and Chronic Effects of cTnC Variants on Cardiac Function

Acute and chronic effects of cTnC variants on cardiac function can beassessed using rAAV6-cTnC vectors and transgenic mice. To determine theresponse to acute changes in myofilament function, normal adult mice canbe transfected via tail vein or intraocular orbit injection ofrAAV6-cTnC variants (e.g., WT, L48Q, L57Q, or 161Q) with a cardiacspecific promoter (cTnT455). Parallel experiments can be performed withthe L48Q cTnC and 161Q cTnC transgenic mice by repressing the MHCpromoter until adulthood. Additional studies can be conducted with micewithout repression of the promoter to determine the effects of thesecTnC variants on normal cardiac development and function.Echocardiographic assessments at 1, 2, 3, and 6 months of age can beconducted to determine onset and progression of any changes in function.Some animals may be stressed via β-adrenergic stimulation withisoproterenol. Following echocardiography, some animals may undergohemodynamic measurements using MILLAR™ catheter protocols, others may beeuthanized and hearts dissected for working heart protocols or forintact or skinned trabeculae preparations, cultured cardiomyocytes, ormyofibril preparations.

Example 8—Tissue-Specific Targeting with rAAV6 Constructs

Tissue specificity was assessed using alkaline phosphatase driven byvarious gene promoters in rAAV6 constructs. Table 2 (see below) comparestwo cardiac specific promoters (creatine kinase 7 (CK7) and cardiactroponin T (cTnT455)) to the non-specific cytomegalovirus (CMV)promoter), with values normalized to CK7 in the TA. cTnT455 can lead tohigh expression in the heart but little to no expression in othertissue. This specificity may reduce potential for effects of R1R2over-expression in non-cardiac tissues.

dATP has no significant effect on mouse aortic smooth muscle forcedevelopment. To study potential systemic effects of elevated dATP it wasdetermined if dATP affects mouse aortic smooth muscle contraction. FIG.12A shows that back to back contractions in skinned muscle strips didnot differ for dATP vs. ATP as the contractile substrate, and the datafor multiple experiments is summarized in FIG. 12B. Additionally,control measurements demonstrated that dATP did not change the level ofmyosin light chain phosphorylation, which controls smooth muscle myosinbinding to actin.

Example 9—Recombinant AAV6-R1R2 for Cardiac-Specific Targeting

rAAV6 vectors were used to acutely increase cardiac levels of R1R2 (and[dATP]). Plasmids can be produced with recombinant rAAV genomescontaining a cardiac specific promoter (cTnT455). R1R2 transgeneexpression cassettes can be co-transfected into HEK293 cells with apackaging/helper plasmid pDGM6 by CaPO₄ precipitation methodology.Vectors can be collected from culture, freeze-thawed, and thesupernatant can be collected. Affinity purification can use a HITRAP™heparin column (GE HEALTHCARE LIFE SCIENCES™, Piscataway, N.J.). Thevector can be concentrated on a sucrose gradient (40%), spun at 27,000rpm (18 hours, 4° C.), and resolubilized in Hanks' balanced solution.Vector genomes can be determined relative to plasmid standards using aSV40 polyadenylation region oligonucleotide ³²P end-labeled probe withSouthern blot hybridization and confirmed by qPCR.

TABLE 2 Comparison of CK7, CMV, and cTnT455 promoters CK7 CMV cTnT455Tibialis Anterior 1 3.1 0 Heart 1.9 5.1 1.6 Lung 0.02 0.09 0.01 Liver0.02 0.09 0.004 Aorta 0.01 0.13 0.005

Selection of the cardiac targeting construct was assessed using alkalinephosphatase driven by various gene promoters in rAAV6 constructs. Table2 compares two striated muscle specific promoters (creatine kinase 7(CK7) and cardiac troponin T (cTnT455)) to the nonspecificcytomegalovirus (CMV) promoter, with values normalized to CK7 in the TA.cTnT455 can lead to high expression in the heart, but little to noexpression in other tissue, thus reducing the potential for effects ofR1R2 over-expression in non-cardiac tissues. FIG. 14A shows Western blotevidence for this, where heart tissue from a rAAV6-RIR2cTnT455 injected(4.5 e¹³) mouse expressed high RI & R2 subunits compared to controlmouse heart. Note that upper bands are nonspecific staining, with arrowspointing to RI and R2 protein (identified by molecular weight markers).R1 & R2 expression in lung was extremely low in comparison with heartand was not changed in skeletal muscle. This is demonstrated in FIG. 14for heart tissue from non-injected (panel “B”) vs. rAAV6-alkalinephosphatase (panel “C”) injected mice, suggesting rAAV6-RIR2cTnT455 mayprovide stable, long-term R1R2 over-expression. Stable rAAV6 transgeneexpression has also been shown to persist for 12 or more weeks in ratand at least 6 or more months in dogs.

Studies may determine the relationship between rAAV6-R1R2cTnT455injection dose, time course, and stability of increased LV pumpfunction, cardiac tissue R1R2 levels, and [dATP]. FIG. 15 shows theeffect of 3 vector doses, i.e., 1.5×10¹³, 4.5×10¹³, and 1.35×10¹⁴rAAV6-RIR2cTnT455 vector genomes or saline (control) injected into 3month old mice (n=6 per group) on LV function. LV fractional shortening(FS) was significantly increased at the high dose after one week and atall doses after two weeks, with equivalent effects by 6 weeks. Themagnitude increase in FS is 25%-50%, indicating the effect that may beachievable with a relatively low vector dose.

Example 10—Transgenic R1R2 Over-Expression Mice (TG-R1R2)

Bi-transgenic mice that over-express both subunits (Rrm1 & Rrm2) of RRcan be utilized. FIG. 13 depicts over-expression of both subunits incardiac muscle, with densitometric calculation values for these TG-R1R2mice that are 33.7±7.6 (Rrm1) and 23.7±3.4 (Rrm2) fold greater thancorresponding values for wild type (WT) mice. Note that for Rrm2 theupper band (*) is non-specific. The endogenous Rrm2 protein is notdetectable in WT tissue, but in TG-R1R2 mice it appears as the bandbelow the background band. While dATP levels for cardiac tissue have notyet been assessed, [dATP] is increased 10-fold in skeletal muscle, whichhad corresponding 3.3±2.1 (Rrm1) and 35.7±11.1 (Rrm2) fold increases inthe enzyme subunits. This magnitude of increase in dATP is similar towhat has been determined for cardiomyocytes transfected withadenovirus-R1R2 in culture (see FIG. 10). Preliminary echocardiographyof these TG-R1R2 mice at 6-8 months of age (measured on 3 successiveweeks) revealed an average >50% increase in fractional shortening (FS)and a 15% reduction in diastolic LV inner diameter (LVIDd). As shown inFIG. 11, these differences (from WT controls) are similar in magnitudeto values for the preliminary adenovirus-R1R2 injection experiments.

Example 11—Acute Effects of Elevated Cellular R1R2 and [dATP] on CardiacFunction

Acute R1R2 over-expression (via rAAV6-R1R2 vectors) may increase [dATP]in mouse hearts, resulting in increased systolic and diastolic function.This may be reflected in: 1) increased cardiomyocyte and myofibrilcontraction with faster relaxation (due in part to increased crossbridgecycling kinetics); 2) an increase in basal cardiac metabolism withoutcompromising energetic reserves; and 3) no change or a decrease inaction potential duration (due to enhanced Ca²⁺ sequestration).

Normal adult FVB/N mice can be transfected via tail vein or intraocularorbit injection with rAAV6-R1R2 vectors with the cardiac specificpromoter cTnT455 (as described above), with sham injections and withrAAV6 containing only cTnT455 as controls. Following injection,echocardiography can be performed weekly (out to 6 weeks) to determinethe optimal (maximal effect) time point for further assessments. Initialstudies may characterize cardiac function in vivo with echocardiography,followed by in situ hemodynamic measures, or ex vivo using Langendorffperfused hearts for energetic studies and a working heart apparatus toassess pump performance. At selected time-points, other mice can beeuthanized and hearts dissected for intact or skinned trabeculaepreparations, isolated cardiomyocytes, myofibril preparations, proteinanalysis, and (immuno)histology. These measurements may providemolecular mechanisms for alterations in cardiac function with acute R1R2over-expression.

Example 12—Myofilament and SR Protein Profiling

Changes in contractile function, Ca²⁺ transients, SR spark activity,and/or Ca²⁺ load under all conditions may be correlated with isoform,abundance, and phosphorylation of myofilament proteins (cTn1, cTnT,MLC-2, cMyBP-C, and Tm), SR proteins (PLB, RyR), and sarcolemmalproteins (NCX, PMCA, and L-type Ca²⁺ channel). Changes in mRNA andprotein expression may be determined using RT-PCR and Western blotanalysis. SR protein fractions can be prepared. If electrophysiologicalmeasurements indicate changes, ion channels can be assessed withspecific antibodies. Analysis of R1R2 expression can be made via Westernblots (see FIG. 10) or immunohistochemistry, and correlated withexperimental endpoints. Specificity of the cTnT455 promoter can beassessed by determining R1R2 expression in non-cardiac tissues such asskeletal muscle and lung. Phosphorylation can be profiled using PRO-Q®Diamond phosphoprotein gel stain (with SYPRO® Ruby Protein Gel Stain)and Western blot analysis. For site specific serine and threonineresidue phosphorylation, mass spectrometry can be performed.

R1R2 over-expression and increased [dATP] may improve cardiacperformance of infarcted hearts at the selected time point for analysis.Response to high Ca²⁺ challenge, β-adrenergic stimulation, andincreasing pre-loads may be improved. In vitro Neely working heartmeasurements of the hearts assessed in FIG. 16 showed a loss of pre-loadresponsiveness of hearts (heart failure) that have been infarcted (notreatment), but showed a recovery of pre-load responsiveness of theinfarcted hearts receiving the vectors to the level of control,uninfarcted hearts, thereby demonstrating a restoration of cardiacfunction. With reference to FIG. 17, where power is given in units ofg·cm/min, the effect may have occurred by lessening chronic β-adrenergicstimulation (which can be assessed by monitoring plasma hormones). Thismay be reflected in the multi-scale analysis as improved: 1) Ca²⁺transients; 2) myofilament contraction and relaxation magnitude andkinetics; and 3) energetic profile. A difference between treated anduntreated hearts in α- and β-adrenergic mediated cardiomyocyte proteinphosphorylation may also be seen.

Example 13—AAV9 Vector Carrying CK8-μDys5

An 8 week old mdx^(4cv) mouse was injected (intramuscularly) into the TAmuscle with 2.5×10¹¹ vector genomes of an AAV9 vector carrying aCK8-μDys5 expression cassette. Two weeks later, the mouse was sacrificedand muscle cryosections were stained for dystrophin expression using ananti-dystrophin antibody. As shown in FIG. 22, widespread and robustexpression of the μDys5 protein was observed in the injected muscle.

An AAV9 vector can comprise an expression cassette (e.g., a promoter, acDNA, and a Poly(A) site) linked to an AAV inverted terminal repeat(ITR). The ITRs may be from AAV2. The AAV9 vector can include thegenomic DNA comprising the expression cassette and the ITR packaged intoa vector using the capsid proteins from AAV9. SEQ ID NO:22 is anexemplary nucleic acid sequence of a CK8-μDys5 cassette with an invertedterminal repeat (ITR) attached. Such a sequence may be used to generateAAV6, AAV9, etc. Different introns, poly(A) sites, spacers, etc. mayalso be added to the sequence.

Example 14—Animal Experiments

Male wild type and dystrophic mdx^(4cv) mice bred on a C57BL/6 inbredstrain were used in this study. Animal experiments were performed inaccordance with the Institutional Animal Care and Use Committee of theUniversity of Washington. For initial screening, 5-6 week-old dystrophicmdx^(4cv) mice were administered 5×10¹⁰ vg of rAAV6 vector into the TAmuscle. Control mice were injected with Hanks' balanced saline solutionas a sham manipulation. In systemic analysis, 14-day old mdx^(4cv) maleswere administered 10¹³ vg of rAAV6 vector intravenously viaretro-orbital injection. Mice were sacrificed at either three or sixmonths post-treatment for further evaluation.

Example 15—Vector Cloning and Virus Production

All micro-dystrophin transgenes were engineered using standard cloningtechniques (see Chamberlain, J., PCR-mediated Mutagenesis,doi:10.1038/npg.els.0003766 (2004)). Modified regions were subclonedinto μDysHinge3 (ΔH2-R23/ΔCT, +H3) within the AAV vector genome backboneplasmid, pARAP4, using MfeI/XhoI or NheI/XhoI restriction sites flankingthe majority of the central rod domain (see Banks, G. B., et al., PLoSGenetics 6, e1000958, (2010)). The polyadenylation signal from therabbit beta-globin gene was subcloned immediately after the μDys cDNAcarboxy terminus. The CMV promoter composed of the cytomegalovirusimmediate early promoter and enhancer drove expression ofmicro-dystrophin cDNA. The CK8 regulatory cassette (see Goncalves, M.A., et al., Molecular Therapy: The Journal of the American Society ofGene Therapy 19, 1331-1341, (2011)) was subcloned in SphI/SaclI sites toreplace the CMV promoter and drive expression of micro-dystrophin cDNAin myogenic cells. Recombinant AAV6 vectors were made as previouslydescribed (see Gregorevic, P., et al., Nature Medicine 12, 787-789,(2006)). Briefly, expression constructs were co-transfected into HEK293cells with pDGM6 packaging plasmid and later harvested and purified by acombination of filtration, heparin affinity chromatography, andultracentrifugation. Viral preparations were quantified by Southern blotand quantitative PCR analysis and always in comparison to otherpreparations used in this study to ensure equal dosing in treatingdystrophic mice.

Example 16—Histological Analysis

After physiological analysis, mice were sacrificed for necropsy. Muscleswere embedded in TISSUE-TEK® O.C.T. Compound, an optimum cuttingtemperature formulation of water-soluble glycols and resins (SAKURAFINETEK USA™, Torrance, Calif.) and frozen in liquid nitrogen-cooledisopentane. Transverse sections approximately 10 μm thick were used forimmunofluorescence studies. Sections were blocked in 2% gelatin and 1%Tween-20 in potassium phosphate buffered saline (KPBS). Sections werewashed with 0.2% gelatin in potassium phosphate buffered saline (KPBS-G)and followed an incubation of primary antibodies diluted in 2% normalgoat serum in KPBS-G. Sections were then rinsed in KPBS-G three timesbefore incubation with secondary antibodies and DAPI,4′,6-Diamidine-2′-phenylindole dihydrochlorid (SIGMA-ALDRICH®, St.Louis, Mo.). After washing three more times in KPBS-G, slides weremounted in PROLONG® GOLD ANTIFADE MOUNTANT, a liquid mountant (LIFETECHNOLOGIES™, Grand Island, N.Y.). Primary antibodies included rabbitpolyclonal N-terminal anti-dystrophin antibody (see Harper, S. Q., etal., Nature Medicine 8, 253-261, (2002)), mouse monoclonalanti-dystrophin (MANEX1011B clone 1C7, Developmental Studies HybridomaBank (DSHB) at the University of Iowa, Iowa City, Iowa) conjugated toALEXA FLUOR® 488 DYE, a green-fluorescent dye (LIFE TECHNOLOGIES™),mouse anti-β-dystroglycan (MANDAG2 clone 7D11, DSHB) conjugated toDYLIGHT™ 594, an amine-reactive dye (THERMO FISHER SCIENTIFIC™,Rockford, Ill.), rat anti-α2-laminin (clone 4H8-2, SIGMA-ALDRICH®, St.Louis, Mo.), and rabbit anti-nNOS (Z-RNN3, LIFE TECHNOLOGIES™).Secondary antibodies were goat anti-rabbit or anti-rat conjugated toALEXA FLUOR® 660 far-red dye or ALEXA FLUOR® 594 red-fluorescent dye,respectively (LIFE TECHNOLOGIES™). Images were captured on an OLYMPUS™SZX16™ dissection fluorescent microscope with DP™ software (OLYMPUS™,Center Valley, Pa.).

Example 17—Immunoblotting

A muscles of mice from an initial screen were snap frozen in liquidnitrogen and then ground by dry ice-chilled mortar and pestle. Muscleswere homogenized in kinase assay lysis buffer (1% Triton X-100, 50 mMTris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA supplemented with COMPLETE™MINI protease inhibitor cocktail tablet (ROCHE™, Indianapolis, Ind.).Protein concentration of lysate was determined using the PIERCE™Coomassie Plus (Bradford) Assay (PIERCE™, Rockford, Ill.). 40 μg ofprotein was suspended in NUPAGE® LDS sample buffer (LIFE TECHNOLOGIES™)supplemented with 100 mM dithiothreitol and loaded onto a NuPAGE® 4-12%Bis-Tris polyacrylamide gel (LIFE TECHNOLOGIES™). After running the gelsand transferring samples onto AMERSHAM™ HYBOND™ P polyvinylidenefluoride membrane (GE HEALTHCARE LIFE SCIENCES™ Piscataway, N.J.), blotswere blocked with 10% nonfat dry milk in PBS. Blots were then incubatedwith primary antibodies in 5% nonfat dry milk, 0.1% Tween-20 in PBS(PBST). After washing three times in PBST, secondary antibodies wereincubated in 5% nonfat milk in PBST and followed by four washes in PBST.Primary antibodies included mouse anti-dystrophin (MANEX1011B clone 1C7,DSHB) and rabbit anti-glyceraldehyde 3-phosphate dehydrogenase (G9545,SIGMA-ALDRICH®) as a loading control. Secondary antibodies includeddonkey anti-rabbit or mouse (JACKSON IMMUNORESEARCH LABORATORIES™, WestGrove, Pa.). Blots were developed with PIERCE™ ECL Plus Western blottingsubstrate (THERMO FISHER SCIENTIFIC™) and scanned using a STORM™ 860imaging system (GE HEALTHCARE LIFE SCIENCES™)

Example 18—Functional Analyses of Skeletal Muscles

Muscles were assayed in situ (gastrocnemius) and in vitro (diaphragm)for force generation and susceptibility to contraction-induced injury aspreviously described with the noted modifications (see Banks, G. B., etal., Human Molecular Genetics 17, 3975-3986, (2008) and Gregorevic, P.,et al., The American Journal of Pathology 161, 2263-2272, (2002)). Themaximum isometric force was determined at optimal muscle fiber lengthand then the muscle was subjected to a series of progressivelyincreasing length changes under stimulation (model 701C™, high-power,bi-phase stimulator, AURORA SCIENTIFIC™). Maximum isometric tetanicforce was measured by stimulating at 150 Hz and 180 Hz for thegastrocnemius and diaphragm, respectively. Eccentric contractions wereperformed at thirty-second intervals, each comprising stimulation at afixed length to allow peak isometric force of either 150 ms(gastrocnemius) or 100 ms (diaphragm), followed by a continued 200 ms(gastrocnemius) or 300 ms (diaphragm) of stimulation during physicallengthening of the muscle. A series of length changes, or strains, of0-45% of the optimum length was applied to potentiate overloading of thecontractile properties and damage to the muscle architecture. The resultfrom an eccentric contraction was measured in the peak isometric forcegenerated just prior to the subsequent eccentric contraction.

Mice were anesthetized with 2,2,2-tribromethanol (SIGMA-ALDRICH®) to beunresponsive to tactile stimuli and then prepped for in situ analysis ofthe gastrocnemius. The Achilles' tendon was exposed by incision at theankle, sutured with 3-0 braided silk (ETHICON™, Cincinnati, Ohio),severed, and secured to the lever arm of a dual-mode forcetransducer-servomotor (model 3058-LR™, AURORA SCIENTIFIC™, Ontario,Calif.). Mice were immobilized and secured to the apparatus by astainless steel pin inserted through the knee, and by taping the hindpaw to a customized PLEXIGLAS®, poly(methyl methacrylate), platform.Gastrocnemius muscle was stimulated via two needle electrodes that wereinserted through the skin on either side of the peroneal nerve in theregion between the knee and hip. The servomotor's position wasmanipulated on three axes to help determine the optimal muscle fiberlength. The servomotor was controlled by LABVIEW™ software that alsoallowed data acquisition (NATIONAL INSTRUMENTS®, Austin, Tex.).

For in vitro preparation of diaphragm, the anesthetized mouse wassacrificed after gastrocnemius analysis and the entire diaphragm muscleand surrounding ribcage was quickly excised to a dish containingoxygenated Tyrode's solution (see Lannergren, J., Bruton, et al., TheJournal of Physiology 526 Pt 3, 597-611 (2000)) containing (mM): NaCl121, KCl 5, CaCl₂) 1.8, MgCl₂ 0.5, NaH₂PO₄ 0.4, NaHCO₃24, glucose 5.5solution as bubbled by 5% CO₂-95% O₂ mixture (pH 7.3). A diaphragm stripcomposed of longitudinally arranged full-length muscle fibers, a portionof the central tendon, and a portion of rib bones and intercostal muscleon the distal end of the strip was isolated under a microscope. Themuscle strip was tied with needle-lead braided surgical silk (6-0, P1;ETHICON™) at the central tendon, sutured through the rib bone portion(5-0; ETHICON™) and then secured to an in situ mouse apparatus with atemperature controlled, horizontal bath (model 809 ATM, AURORASCIENTIFIC™). Apparatus bath was filled with the bubbled Tryode'ssolution described above and maintained at 25° C. Optimal fiber lengthwas determined and isometric and eccentric contractile properties wereassessed in a manner similar to gastrocnemius muscle analysis, with theconditions specified above for the diaphragm muscle. Specific force ofboth muscle groups was determined by normalizing maximum isometric forceto the mass of the gastrocnemius muscle or diaphragm strip,respectively. The following equation was used: specific force=maximumforce×pennation×muscle length×1.04 density/muscle weight (seeBurkholder, T. J., et al., Journal of Morphology 221, 177-190, (1994)).Pennation is the angle at which bundles of skeletal muscle fibers orientthemselves between the tendons of the muscle. For the gastrocnemiusmuscle, this angle was determined by a previous study (see Banks, G. B.,et al., PLoS Genetics 6, e1000958, (2010)). Diaphragm muscle strips wereisolated in such a way that the myofibers would contract in a directline between the semitendinosus junction to the myotendinous junction atthe rib (see Gregorevic, P., et al., The American Journal of Pathology161, 2263-2272, (2002)). Pennation for the gastrocnemius and diaphragmequals 0.45 and 1, respectively.

Example 19—Construction of the cTnT455 Regulatory Cassette

The cTnT455 regulatory cassette (SEQ ID NO:1; 455 indicates the numberof base pairs in the RC) was constructed as described herein. DNA wasprepared from human cells. PCR primers were used to amplify the cTnTenhancer/promoter region based on sequence similarity to rat and chickencTnT sequences. The wildtype cTnT enhancer/promoter was ligated to ahuman placental alkaline phosphatase (AP) cDNA, and plasmid DNA wasproduced. cTnT-AP plasmids were transfected into newborn ratcardiomyocytes and into differentiating mouse skeletal muscle cells.

The wild type human cTnT RC (SEQ ID NO:2) had high activity in bothcardiac and skeletal muscle cell cultures. cTnT's expression in skeletalmuscle was initially unanticipated. Without being bound by any oneparticular theory, however, cTnT expression in skeletal muscle may bedue to the normal activation of cardiac gene expression during earlyskeletal muscle development and during muscle regeneration. Thisproperty may be potentially beneficial for some gene therapyapplications, for example, such as in the transient expression of atherapeutic protein only during muscle regeneration.

The wild type cTnT enhancer was then miniaturized by removingnon-conserved base sequences (based on comparisons between human, rat,dog, and chicken) as well as some conserved sequence motifs, followed bytransfection tests, as discussed above, to verify that the deletions didnot decrease transcriptional activity (see FIGS. 18 and 19)

To obtain higher activity, it was tested whether the addition ofmultiple miniaturized cTnT enhancers to the cTnT promoter would increaseactivity. These tests were carried out in cardiac and skeletal musclecultures and cTnT455 (containing one extra enhancer) was found to be themost active (see FIG. 20).

To determine whether cTnT455 was active in vivo, the cTnT455-APconstruct was packaged in rAAV6, and the vectors were administered viaretro-orbital systemic delivery to mice. Four weeks later, the mice wereeuthanized and assays were carried out for RC expression levels incardiac as well as skeletal muscles and non-muscle tissues. The datashowed that cTnT455 had high transcriptional activity in cardiac muscleand was transcriptionally silent in both skeletal muscles and allnon-muscle tissues (see Table 2; see also, PCT Application No.PCT/US2012/039897 entitled “Cell and Gene Based Methods to ImproveCardiac Function”, the entirety of which is incorporated by referenceherein).

Example 20—Statistical Analysis

All results are reported as mean±standard error mean. Differencesbetween cohorts were determined using one-way and two-way ANOVA withTukey's post hoc multiple comparison test. All data analyses wereperformed with GRAPHPAD™ PRISM™ 6 software (San Diego, Calif.).

As will be understood by one of ordinary skill in the art, eachembodiment disclosed herein can comprise, consist essentially of, orconsist of its particular stated element, step, ingredient, orcomponent. As used herein, the transition term “comprise” or “comprises”means includes, but is not limited to, and allows for the inclusion ofunspecified elements, steps, ingredients, or components, even in majoramounts. The transitional phrase “consisting of” excludes any element,step, ingredient or component not specified. The transition phrase“consisting essentially of” limits the scope of the embodiment to thespecified elements, steps, ingredients or components, and to those thatdo not materially affect the embodiment.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,and so forth used in the specification and claims are to be understoodas being modified in all instances by the term “about.” Accordingly,unless indicated to the contrary, the numerical parameters set forth inthe specification and attached claims are approximations that may varydepending upon the desired properties sought to be obtained by thepresent invention. At the very least, and not as an attempt to limit theapplication of the doctrine of equivalents to the scope of the claims,each numerical parameter should at least be construed in light of thenumber of reported significant digits and by applying ordinary roundingtechniques. When further clarity is required, the term “about” has themeaning reasonably ascribed to it by a person skilled in the art whenused in conjunction with a stated numerical value or range, i.e.,denoting somewhat more or somewhat less than the stated value or range,to within a range of ±20% of the stated value; ±19% of the stated value;±18% of the stated value; ±17% of the stated value; ±16% of the statedvalue; ±15% of the stated value; ±14% of the stated value; ±13% of thestated value; ±12% of the stated value; ±11% of the stated value; ±10%of the stated value; ±9% of the stated value; ±8% of the stated value;±7% of the stated value; ±6% of the stated value; ±5% of the statedvalue; ±4% of the stated value; ±3% of the stated value; ±2% of thestated value; or ±1% of the stated value.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements.

The terms “a,” “an,” “the” and similar referents used in the context ofdescribing the invention (especially in the context of the followingclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.Recitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention otherwise claimed. No languagein the specification should be construed as indicating any non-claimedelement essential to the practice of the invention.

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember may be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. It isanticipated that one or more members of a group may be included in, ordeleted from, a group for reasons of convenience and/or patentability.When any such inclusion or deletion occurs, the specification is deemedto contain the group as modified thus fulfilling the written descriptionof all Markush groups used in the appended claims.

Certain embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention. Ofcourse, variations on these described embodiments will become apparentto those of ordinary skill in the art upon reading the foregoingdescription. The applicants expect skilled artisans to employ suchvariations as appropriate, and the applicants intend for the variousembodiments of the disclosure to be practiced otherwise thanspecifically described herein. Accordingly, this disclosure includes allmodifications and equivalents of the subject matter recited in theclaims appended hereto as permitted by applicable law. Moreover, anycombination of the above-described elements in all possible variationsthereof is encompassed by the disclosure unless otherwise indicatedherein or otherwise clearly contradicted by context.

Furthermore, numerous references have been made to patents and printedpublications throughout this specification. Each of the above-citedreferences and printed publications are individually incorporated hereinby reference in their entirety.

It is to be understood that the embodiments of the present disclosureare illustrative of the principles of the present disclosure. Othermodifications that may be employed are within the scope of thedisclosure. Thus, by way of example, but not of limitation, alternativeconfigurations of the present disclosure may be utilized in accordancewith the teachings herein. Accordingly, the present disclosure is notlimited to that precisely as shown and described.

The particulars shown herein are by way of example and for purposes ofillustrative discussion of the preferred embodiments of the presentdisclosure only and are presented in the cause of providing what isbelieved to be the most useful and readily understood description of theprinciples and conceptual aspects of various embodiments of thedisclosure.

Definitions and explanations used in the present disclosure are meantand intended to be controlling in any future construction unless clearlyand unambiguously modified in the examples or when application of themeaning renders any construction meaningless or essentially meaninglessin cases where the construction of the term would render it meaninglessor essentially meaningless, the definition should be taken fromWebster's Dictionary, 3rd Edition or a dictionary known to those ofordinary skill in the art, such as the Oxford Dictionary of Biochemistryand Molecular Biology (Ed. Anthony Smith, Oxford University Press,Oxford, 2004).

It will be apparent to those having skill in the art that many changesmay be made to the details of the above-described embodiments withoutdeparting from the underlying principles of the invention. The scope ofthe present invention should, therefore, be determined only by thefollowing claims.

1. A pharmaceutical composition, the composition comprising: amicro-dystrophin gene encoding a protein comprising: an amino-terminalactin-binding domain; a β-dystroglycan binding domain; and aspectrin-like repeat domain consisting of five spectrin-like repeats,including spectrin-like repeat 1 (SR1), spectrin-like repeat 16 (SR16),spectrin-like repeat 17 (SR17), spectrin-like repeat 23 (SR23), andspectrin-like repeat 24 (SR24); wherein the micro-dystrophin gene isoperatively linked to a regulatory cassette; and a delivery vehicle,wherein the pharmaceutical composition is formulated for intravenous orintramuscular administration.
 2. The pharmaceutical composition of claim1, wherein the protein encoded by the micro-dystrophin gene furthercomprises at least a portion of a hinge domain.
 3. The pharmaceuticalcomposition of claim 2, wherein the hinge domain is selected from atleast one of a Hinge 1 domain, a Hinge 2 domain, a Hinge 3 domain, and aHinge 4 domain.
 4. The pharmaceutical composition of claim 1, whereinthe regulatory cassette is selected from the group consisting of a CK8promoter and a cardiac troponin T (cTnT) promoter.
 5. The pharmaceuticalcomposition of claim 4, wherein the regulatory cassette is configured toexpress the micro-dystrophin gene such that a level of expression of themicro-dystrophin gene is at least about 100-fold higher in striatedmuscle cells than the level of expression of the micro-dystrophin genein non-muscle cells.
 6. The pharmaceutical composition of claim 4,wherein the regulatory cassette is a CK8 promoter, and wherein the CK8promoter has at least 80% sequence identity to the nucleic acid sequenceof SEQ ID NO:19.
 7. The pharmaceutical composition of claim 1, whereinthe delivery vehicle comprises an adeno-associated virus (AAV) vector ora recombinant adeno-associated virus (rAAV) vector.
 8. Thepharmaceutical composition of claim 7, wherein the adeno-associatedvirus (AAV) vector consists of serotype AAV6, AAV8, or AAV9.
 9. Thepharmaceutical composition of claim 7, wherein the recombinantadeno-associated virus (rAAV) vector consists of rAAV6, rAAV8, rAAV9, orrAAV2/6.
 10. The pharmaceutical composition of claim 1, wherein thepharmaceutical composition is formulated for intramuscularadministration.
 11. The pharmaceutical composition of claim 1, whereinthe pharmaceutical composition is formulated for intravenousadministration.
 12. The pharmaceutical composition of claim 1, whereinpharmaceutical composition is formulated to reduce at least onepathological effect or symptom of a muscular dystrophy.
 13. Thepharmaceutical composition of claim 12, wherein the muscular dystrophyis selected from the group consisting of: Duchenne muscular dystrophy,Becker muscular dystrophy, limb girdle muscular dystrophy,fascioscapulohumeral muscular dystrophy, congenital muscular dystrophy,oculopharyngeal muscular dystrophy, Emery-Dreifuss muscular dystrophy,and myotonic muscular dystrophy.
 14. The pharmaceutical composition ofclaim 12, wherein the muscular dystrophy is Duchenne muscular dystrophy.15. The pharmaceutical composition of claim 12, wherein the musculardystrophy is Becker muscular dystrophy.
 16. The pharmaceuticalcomposition of claim 12, wherein the pathological effect or symptom isselected from the group consisting of: muscle pain, muscle weakness,muscle fatigue, muscle atrophy, fibrosis, inflammation, increase inaverage myofiber diameter in skeletal muscle, cardiomyopathy, reduced6-minute walk test time, loss of ambulation, and cardiac pump failure.